UNC5B is a known tumor suppressor gene in a variety of cancers. As a transmembrane protein, UNC5B also induces apoptosis in a P53-dependent manner. In this study, we demonstrate that UNC5B inhibits proliferation through G2/M phase arrest by mass spectrometry and bioinformatics analysis in bladder cancer cells. By combing with CDC14A and P53, UNC5B dephosphorylated P53 at Ser-315 site. This dephosphorylation facilitated G2/M phase arrest by reducing the expression of cyclin B1 and increasing the expression of p-CDK1, thus inhibiting tumor proliferation. Knockdown of CDC14A suppressed the G2/M phase arrest induced by UNC5B in vitro, and eliminated the inhibitory effect of UNC5B on tumor proliferation in vivo. Our results show that UNC5B-mediated cell cycle arrest may act as a potential treatment for bladder cancer.