We previously showed that the zinc finger-containing transcription factor Krüppel-like factor 5 (KLF5) is important in mediating transformation by oncogenic H-Ras through induction of cyclin D1 expression and acceleration of the G1/S transition of the cell cycle. Here we present evidence of a role for KLF5 in accelerating mitotic entry in H-Ras-transformed NIH3T3 fibroblasts. When compared with non-transformed parental NIH3T3 cells, H-Ras-transformed fibroblasts exhibit an increase in mitotic index, levels of cyclin B1 and Cdc2, and cyclin B1/Cdc2 kinase activity. Inhibition of KLF5 expression in H-Ras-transformed cells with KLF5-specific small interfering RNA (siRNA) results in a decrease in each of the aforementioned parameters, with a concomitant reduction in the transforming potential of the cells. Conversely, over-expression of KLF5 in NIH3T3 cells leads to an increase in the promoter activity of the genes encoding cyclin B1 and Cdc2. These results indicate that KLF5 accelerates mitotic entry in H-Ras-transformed cells by transcriptionally activating cyclin B1 and Cdc2, which leads to an increase in cyclin B1/Cdc2 kinase activity. Extending our previous observation that KLF5 activates cyclin D1 transcription to promote G1/S transition, our current results further support a crucial function for KLF5 in mediating cellular transformation caused by oncogenic H-Ras.