The calcium-binding protein S100A4 induces the metastatic phenotype in rodent models of breast cancer, and its expression strongly correlates with reduced survival in human breast and bladder cancer. We have established an orthotopic model of bladder cancer by injecting a cell line derived from a carcinogen-induced rat bladder tumor into the muscular wall of syngeneic rats. MYU-3L cells produce rapidly growing, invasive tumors in the bladder wall but they fail to metastasize. Transfection of MYU-3L cells with a plasmid vector directing overexpression of the S100A4 gene generates variants in which S100A4 expression is elevated by up to sevenfold in comparison with the untransfected cells. Variants overexpressing S100A4 produce primary tumors at similar frequencies and latencies to the parental cell line, a significant number of which metastasize to the para-aortic lymph nodes or lungs. Expression of S100A4 protein in the primary tumors was heterogeneous, but was stronger and more consistent in the metastases, suggesting that transfectants overexpressing S100A4 possess an enhanced ability to form metastatic lesions. We conclude that overexpression of S100A4 can induce the metastatic phenotype in this rodent model of bladder cancer. Taken together with the results from our parallel studies of human bladder cancer, these data suggest a significant role for S100A4 in bladder cancer metastasis and identify a potential new target for systemic therapy in patients with this disease.