Technical Abstract: The ubiquitin-proteasome pathway plays an important role in controlling the cell cycle. The purpose of this study was to examine if expression of a dominant negative form of ubiquitin can inhibit the proliferation of lens epithelial cells. Dominant negative K6W-ubiquitin was expressed in cultured human lens epithelial cells via an adenoviral vector. Cell proliferation was monitored by both cell counting and flow cytometry analysis. Protein levels of cell cycle regulators were assessed by western blotting. Expression of K6W-ubiquitin in lens epithelial cells prevented cell proliferation and specifically caused cell cycle delay/arrest in the G2/M phase. Consistent with the cell cycle delay/arrest in the G2/M phase, typical substrates of the ubiquitin-proteasome pathway and also M phase regulators such as cyclin A, cyclin B, and securin were stabilized by expression of K6W-ubiquitin. Cell cycle-dependent degradation of G1 phase regulators, such as the Cdk inhibitor p27KIP, was also inhibited by the expression of K6W-ubiquitin. These data demonstrate that the ubiquitin proteasome pathway plays an important role in regulating lens epithelial cell proliferation. Expression of dominant negative K6W-ubiquitin inhibits lens cell proliferation by inhibiting the degradation of cell cycle regulators.