Diacylglycerol kinases (DGKs) are key regulators of diacylglycerol-dependent signaling pathways. Among the 10 DGK isoforms, DGK-zeta is the only nuclear form that contains a nuclear localization signal. Here, by site-directed mutagenesis, we showed that DGK-zeta also displays a functional independent nuclear export signal (NES) sequence between the amino acid residues 362-370. Indeed, the NES mutant forms of DGK-zeta accumulated in the nucleus to a much greater extent than wildtype DGK-zeta. Moreover, treatment with leptomycin B, an inhibitor of leucine-rich type NES, resulted in accumulation of both endogenous and ectopically expressed DGK-zeta in the nucleus, demonstrating that nuclear export of DGK-zeta is chromosome regional maintenance protein 1 (CRM1)-dependent. Previously, we reported that nuclear DGK-zeta is a negative regulator of cell cycle progression in C2C12 mouse myoblasts. In this paper, we documented that enhancement of DGK-zeta nuclear localization by NES sequence mutation, increases G(0)/G(1) block in C2C12 cells. Overall, our data demonstrate that DGK-zeta export from nucleus to cytoplasm is regulated by a leucine-rich NES through the exportin CRM1 and suggest that the nuclear localization of DGK-zeta could finely tune its function as a regulator of G(1)/S cell cycle transition.