The cell commits to dividing during the G2-M transition, and timing of mitotic entry must be tightly regulated to ensure correct chromosome segregation. Identification of all proteins and molecular events that orchestrate the G2-M transition will be required for a complete understanding of the cell division cycle, and how its deregulation contributes to cell transformation. We have previously reported D53, a member of the tumor protein D52 family, to be a novel 14-3-3 partner protein in breast cancer cells. We now report that D53 expression is highly upregulated at the G2-M transition in breast cancer cell lines in which D53 is endogenously or exogenously expressed. The timing and subcellular localization of D53 expression paralleled that of cyclin B1, and D53 expression was similarly regulated at both post-transcriptional and post-translational levels. Interactions between D53 and 14-3-3, a negative regulator of the G2-M transition, were increased in synchronized populations enriched for cells in G2/M phases, compared with G1/S arrested cells. Enforced expression of two EGFP-tagged D53 isoforms and the related protein D52 produced high proportions of multinucleated MDA-MB-231 breast carcinoma cells. These results identify D53 as a cell cycle-regulated protein whose deregulated expression can adversely affect the completion of mitosis.