Abstract The cyclin E–cyclin-dependent kinase 2 (CDK2) complex accelerates entry into the S phase of the cell cycle and promotes polyploidy, which may contribute to genomic instability in cancer cells. The effect of low molecular weight isoforms of cyclin E (LMW-E) overexpression on mitotic progression and its link to genomic instability were the focus of this study. Here, we show that full-length cyclin E (EL) and LMW-E overexpression impairs the G2-M transition differently by targeting dual-specificity phosphatase Cdc25C activity. We identify Cdc25C as an interaction partner and substrate for cyclin E/CDK2 kinase. Specifically, the cyclin E/CDK2 complex phosphorylates Cdc25C on Ser214, leading to its premature activation, which coincides with higher cyclin B/CDK1 and Polo-like kinase 1 (PLK1) activities in an S-phase–enriched population that result in faster mitotic entry. Whereas EL overexpression leads to hyperactivation of Cdc25C, cyclin B/CDK1, and PLK1 in a G2-M–enriched population, LMW-E overexpression causes premature inactivation of Cdc25C and PLK1, leading to faster mitotic exit. In addition, LMW-E–overexpressing cells showed a reduction in the mitotic index in the presence of a spindle poison and faster degradation of cyclin B, suggesting an increased rate of mitotic slippage and adaptation to the spindle checkpoint. Lastly, downregulation of Cdc25C inhibits LMW-E–mediated chromosome missegregation, anaphase bridges, and centrosome amplification. These results suggest that the high levels of LMW-E isoforms found in breast cancer may contribute to cellular transformation and genomic instability by impairing mitotic progression involving Cdc25C. Cancer Res; 70(12); 5085–95. ©2010 AACR.