Multiple genetic alterations are attributed to gastric cancer (GC); however, only a few critical genes have been identified so far. In this study, we isolated and characterized a novel gene p42.3, represented as tumor-specific and mitosis phase-dependent expression protein in GC cell line BGC823. Our data showed that the expression of p42.3 was cell cycle-dependent in GC cell lines. Moreover, p42.3 was specifically expressed in primary GC tissues but not in the matched normal mucosa of stomach, and this gene was expressed in diverse embryonic tissues. Furthermore, significant suppression of cell proliferation and tumorigenicity were detected and G(2)/M phase arrest was observed in cell line BGC823 depleted of p42.3 expression by RNAi technique, and we confirmed the expression changes of cyclin B1 and Chk2 following the silence of p42.3. Taken together, we cloned and characterized p42.3 gene that was specifically expressed in GC tumors but not in normal gastric mucosa, and the gene was associated with M-phase regulation. Moreover, p42.3 might be involved in cell proliferation and tumorigenesis; therefore, this gene might have potential applications in the diagnosis or treatment of GC.