CXCL12/CXCR4 signaling plays important roles in tumor cell metastasis in many types of cancers, and CXCR4 is the key regulator of cell motility in bladder cancer. Emerging evidence suggests that transcription-3 (Stat3) activation is associated with bladder cancer cell growth and survival, while the relationship between CXCL12/CXCR4 signal and Stat3 activation remains unclear. In this study, expression analysis of bladder cancer and adjacent normal tissues showed that higher CXCR4 expression was associated with Stat3 phosphorylation. CXCR4 knockdown in bladder cancer T24 cells impaired CXCL12-induced cell invasion and Stat3 activation. Furthermore, blocking Stat3 activity with the chemical inhibitor Stattic inhibited CXCL12-triggered Stat3 phosphorylation and cell invasion in T24 cells, suggesting that Stat3 activation is required for CXCL12 function in the mobility of bladder cancer. Taken together, CXCR4 is necessary for CXCL12 signal transduction in bladder cancer, and CXCL12/CXCR4 promotes invasion of bladder cancer cells through activation of Stat3 transcriptional activity.