Ionic zinc (Zn2+) is an essential micronutrient that plays important roles in maintaining cellular and organismal health. As a cofactor in catalysis and secondary messenger in signaling, Zn2+ plays a central but understudied role in a myriad of cellular processes including proliferation and genome stability. We are still unsure how depletion of cellular Zn2+ acts to impair proliferation. To address this, I used high content imaging and flow cytometry of live and fixed cells and found that short exposure to ZD impairs DNA synthesis and leads to simultaneous activation of replication stress signaling. I then identified that Zn2+ is required for S-phase progression, and depletion of Zn2+ during S-phase will elongate mother cell S-phase, as well as cause transient quiescence in daughter cells after division. This quiescence is maintained by sustained expression of the tumor suppressor p21. I also examined changes in free Zn2+ throughout the cell cycle. Using a genetically encoded Förster resonance energy transfer (FRET) based sensor for measuring labile Zn2+, my colleagues and I found that cells experience a transient increase in labile Zn2+ in early G1, which we named the Zn2+ pulse, that scaled with available media Zn2+. Knockdown of the metal responsive transcription factor MTF-1 with an shRNA impaired reestablishment of Zn2+ levels after perturbation. Further, knockdown of MTF-1 increased resting free Zn2+ by impairing expression of metallothionein. Finally, knockdown of MTF-1 decreased cell proliferation in high Zn2+ using both a bulk assay and single-cell microscopy. Together, this work sheds new light on Zn2+ regulation and fluctuations in the mammalian cell cycle.