Human urinary bladder cancer (UBC) is the fourth most common cancer and the eighth most common cause of cancer death in the USA. High mobility group box 3 (HMGB3), a member of a family of proteins containing one or more high mobility group DNA binding motifs, was reported to be overexpressed in a variety of human cancers. However, the expression and role of HMGB3 in human UBC remains unclear. Here, we found that UBC patients had upregulated HMGB at both mRNA and protein levels. Immunochemistry (IHC) evaluation of HMGB3 expression in 113 UBC clinical specimens showed that high expression of HMGB3 had positive correlation with UBC tumor size (P = 0.019), tumor WHO grade (P = 0.031), stage (P = 0.028), and lymph node metastasis (P = 0.017). Moreover, patients with higher HMGB3 expression showed a poorer overall survival rate than those with relatively low HMGB3 (P = 0.0079, log-rank test). Multivariate analysis revealed that HMGB3 expression is an independent prognostic marker. The UBC cancer cell proliferation and migration ability were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and wound healing assays, respectively. RNA interference of HMGB3 in UBC cell lines inhibited cancer cell growth and migration, along with the downregulation of PCNA and MMP2 protein levels. In sum, our data suggests HMGB3 may serve as an important oncoprotein and indicate that overexpression of HMGB3 in UBC could be used as a potential prognostic marker.