The high-mobility group nucleosome-binding domain 5 (HMGN5) is a new and typical member of HMGN protein family. Numerous studies confirmed that HMGN5 was highly expressed in several kinds of malignant tumors, but its role in cancer progression of urothelial bladder cancer (UBC) has not been fully clarified. This study aimed to further investigate the oncogenic role of HMGN5 in UBC 5637 cells employing in vitro and in vivo models and to explore the mechanism [corrected].RNA interference was used to down-regulate HMGN5 expression in 5637 cells by a shRNA expression lentiviral vector. Then cell viability, apoptosis and cell cycle distribution, invasion were detected by MTT assay, flow cytometry and transwell assay, respectively. Tumor growth was also evaluated in nude mice. As a result, successful transfection was confirmed using fluorescence microscopy and HMGN5 was efficiently inhibited. HMGN5 knockdown suppressed invasion, and induced G1/S cell cycle arrestbut not apoptosis and thus contributed to decreased cell viability in UBC 5637 cells [corrected]. Consistent with the cell cycle arrest, the protein expression levels of cyclin D1 were decreased. In vivo study further showed that HMGN5 knockdown affected the tumorigenesis of 5637 cells in nude mice. Western blot also demonstrated that the expression of E-cadherin was enhanced, while the expression of VEGF-C was decreased in 5637 cells depleted of HMGN5 [corrected].In conclusion, we provide both in vivo and in vitro evidence that HMGN5 contribute to the growth and invasion of UBC 5637 cell line and HMGN5 could be exploited as a target for therapy in UBC.