The aim of the present study was to investigate the expression and potential roles of CD74 in human urothelial cell carcinoma of the bladder (UCB) in vitro and in vivo. CD74 and macrophage migration inhibitory factor (MIF) were located and assayed in normal and UCB samples and cell lines using immunostaining. CD74 was knocked down using CD74 shRNA lentiviral particles in HT-1376 cells. The proliferative, invasive potential and microvessel density (MVD) of knockdown-CD74 HT-1376 cells were analyzed in vitro or in vivo. The expression of CD74 in an additional high grade UCB J82 cell line was also verified in vivo. All experiments were repeated at least 3 times. The majority of muscle-invasive bladder cancer (MIBC) samples, and only one high grade UCB cell line, HT-1376, expressed CD74, compared with normal, non-muscle-invasive bladder cancer (NMIBC) samples and other cell lines. The levels of proliferation and invasion were decreased in the CD74 knockdown-HT-1376 cells, and western blotting assay indicated that the levels of proteins associated with proliferation, apoptosis and invasion in the cells were affected correspondingly by different treatments in vitro. The tumorigenesis and MVD assays indicated less proliferation and angiogenesis in the knockdown-HT-1376 cells compared with the scramble cells. Notably, J82 cells exhibiting no signal of CD74 in vitro presented the expression of CD74 in vivo. The present study revealed the potential roles of CD74 in the proliferation, invasion and angiogenesis of MIBC, and that it may serve as a potential therapeutic target for UCB, but additional studies are required.