R. Moses ben Mordecai Zacuto (c. 1620–1697) was born into a Portuguese Marrano family in Amsterdam. He studied Jewish subjects under R. Saul Levi Morteira (an elegy on the latter's death by R. Zacuto was published by D. Kaufmann in REJ 37 (1898), 115). He also studied secular subjects. According to tradition, he later fasted 40 days "in order to forget the Latin language." R. Zacuto was a student in the bet midrash of Amsterdam and in his youth traveled to Poland to study in the yeshivot there. There he was attracted by Kabbalah and refers in his letters to his teacher R. Elhanan, perhaps "R. Elhanan the kabbalist," who died in Vienna in 1651. R. Zacuto moved to Italy, remaining for some time in Verona. From 1645 he lived in Venice and served for a time as a preacher under R. Azariah Figo. Afterward, he became one of the rabbis of the city and a member of the Venetian yeshiva. Between 1649 and 1670 he was proofreader of many books printed in Venice, especially works on Kabbalah. He edited the Zohar Hadash in 1658, and also wrote many poems for celebrations and special occasions. R. Zacuto tried to acquire the manuscripts of the Safed kabbalists, especially those of R. Moses Cordovero and the different versions of the works of R. Hayyim Vital. He befriended the kabbalist R. Nathan Shapiro of Jerusalem and the old kabbalist R. Benjamin ha-Levi, who served as an emissary from Safed in Venice for two years (1658–59).

At the outset of the Shabbatean movement, Zacuto tended to give credence to the messianic tidings, but he was opposed to innovations such as abolition of tikkun hazot ("midnight prayers") and other customs. In the spring of 1666, in a letter to R. Samson Bachi, he took a positive but cautious stand in favor of the movement, mainly supporting its advocacy of repentance. After the apostasy of Shabbetai Zevi he turned his back on the movement and joined the other Venetian rabbis in their action against Nathan of Gaza when he came to Venice in the spring of 1668. In 1671 he was invited to serve as rabbi in Mantua, but he did not go until 1673, remaining there until his death. He enjoyed great authority as the chief of the contemporary Italian kabbalists and corresponded with kabbalists in many places. He never realized his desire to settle in Erez Israel.

R. Zacuto's published exoteric works include his commentary on the Mishnah, Kol ha-Re-Me-Z. A collection of halakhic responsa was published in Venice in 1760. A commentary on the Palestinian Talmud is lost. His major activity, however, was in Kabbalah. R. Zacuto opposed the mingling of the kabbalistic system of R. Cordovero with that of R. Isaac Luria which was then current in some circles. He went over the entire corpus of R. Luria's and R. Vital's writings and added many annotations under the name Kol ha-Re-Me-Z or the abbreviation Ma-Za-La-N. Many of them are collected in the books Mekom Binah and Sha'arei Binah of R. Isaac Sabba (Salonika, 1812–13) and they have partly also appeared in different editions of the works of R. Vital and R. Jacob Zemah. R. Zacuto wrote at least two commentaries on the Zohar. The printed part contains the commentary up to Zohar I, 147b (Venice, 1663). For unknown reasons it was never circulated. Mikdash ha-Shem, his second commentary on the Zohar, was written for the most part according to the Lurianic Kabbalah, and was published in abridged form in the Mikdash Melekh of R. Shalom Buzaglo. R. Zacuto arranged tikkunim ("special prayers") for several religious ceremonies according to Kabbalah. These were often reprinted and had great influence, especially on the religious life in Italy. They include Sefer ha-Tikkunim (a tikkun for the eve of Shavuot and Hoshana Rabba; Venice, 1659), Mishmeret ha-Hodesh (ibid., 1660), Tikkun Shovavim (the initials of the first six sections of Exodus), i.e., a tikkun for fasts undertaken in expiation for nocturnal ejaculations (ibid., 1673), and Tikkun Hazot (ibid., 1704). All these were arranged under the influence of R. Benjamin ha-Levi and R. Nathan Shapiro.

A major part of R. Zacuto's poetry is devoted to kabbalistic subjects, such as his poems in the book Hen Kol Hadash (Amsterdam, 1712), in Tofteh Arukh (a description of hell; Venice, 1715). Besides this he arranged voluminous collectanea on kabbalistic subjects. The first was Shibbolet shel Leket, on all the books of the Bible. This was followed by Remez ha-Romez on numbers, gematria, and explanations of Holy Names according to numerology; and many others.