The aim of the present study was to investigate the expression of the chemokine receptor CXCR4 in human bladder carcinoma cells and its possible role in their metastatic growth in vitro. Immunofluorescent imaging and flow cytometry were performed to examine the expression of CXCR4 in the EJ and EJ-M3 cell lines. A highly specific CXCR4 antagonist, AMD3100, was used to inhibit metastatic growth, as assessed by an in vitro invasion assay. The expression of CXCR4 protein was mainly observed in the cytoplasm of the two bladder carcinoma cell sublines. CXCR4 expression was increased in the EJ-M3 cell line compared to the EJ cells. Differences in the CXCR4 protein expression rates between the EJ-M3 cell line (55.7 ± 10.35%) and EJ cell line (25.4 ± 4.69%) were significant (P < 0.05). The CXCR4 antagonist AMD3100 partially inhibited the metastasis of the EJ-M3 cells, which had higher CXCR4 expression (56 ± 8.96) than the control EJ cells (118 ± 6.56) (P < 0.05). In conclusion, the high in vitro metastatic potential of human bladder carcinoma cell sublines is closely associated with increased CXCR4 expression, and is inhibited by a specific CXCR4 antagonist.