In an earlier study, we cloned the p42.3 gene and showed that its expression was specific to tumors in a number of tumor cell lines and primary tumor tissues. However, the biological role and function of this gene remains largely unknown. In this study, p42.3 expression was found to be cell cycle-dependent at both the mRNA and protein levels in several human tumor cell lines. Typically, abundant expression was detected at G1 and M phases compared with S and G2 phases. Expression peaked at early G1 phase then decreased drastically at late G1, S, and G2. Furthermore, transfection of the p42.3 gene into NIH3T3 cells promoted malignant transformation, accompanied by accelerated mitotic progression and altered chromosome segregation. It was also observed that Cyclin B1 was upregulated and Cdc2-Tyr15 was downregulated following p42.3 overexpression in NIH3T3 cells. Combined, these results indicate that p42.3 as a cell cycle-regulated gene contributes to promoting cell cycle progression through disruption of mitotic regulation, and may play important roles in malignant transformation. © 2012 Wiley Periodicals, Inc.