Increasing β-cell mass is a crucial therapeutic target for diabetes mellitus. Our previous study showed that diacylglycerol kinase δ (DGKδ) is localized in the nucleus and acts as a suppressor of β-cell proliferation. DGKζ, a type IV DGK isoform, has been shown to localize in the nucleus of rat β-cells and be a negative regulator of cell cycle in the fibroblasts. We therefore examined the possibility that DGKζ, similarly to DGKδ, regulates β-cell mass. The intracellular localization of DGKζ was examined in MIN6 cells, a mouse β-cell line, and in isolated mouse islets by subcellular fractionation. Insulin secretion was measured by batch incubation and cell cycle was analyzed by flow cytometry. In MIN6 cells, DGKζ was localized in the cytoplasm and knockdown of DGKζ caused defective insulin secretion but did not affect cell cycle. In contrast, DGKζ in isolated islets was detected in the nucleus fraction as well as in the cytoplasm fraction. EGFP-tagged DGKζ overexpressed in MIN6 cells was also distributed in the nucleus. Moreover, overexpression of DGKζ induced cell cycle arrest in MIN6 cells. These results suggest that cytoplasmic DGKζ regulates insulin secretion, whereas DGKζ expressed in the nucleus regulates cell cycle. Thus, DGKζ is likely to have different function depending on its localization in pancreatic β-cells.