The aim of this study is to investigate the expression of CXCR4 receptor in cervical adenocarcinoma and related mechanisms involved in pelvic lymph node metastasis. Immunohistochemistry was used to evaluate the expression of CXCR4 and the association of pelvic lymph node metastasis in archived tissue from clinical stage IB cervical adenocarcinomas (n = 37) and from benign specimens obtained at hysterectomy for other causes (n = 48). The HeLa cell (cervical adenocarcinoma-derived cell) line that expresses CXCR4 was used to study the interaction between the CXCR4 receptor and stromal cell-derived factor 1alpha (SDF-1alpha). Cell migration assays, cell numbers, flow cytometry, cell proliferation assay, and western blot were used to study the function of CXCR4 and its downstream signal transduction. The positive cases were semiquantitatively divided into three score classes according to their staining. Tumors with strong CXCR4 stainings were more likely to have pelvic lymph node metastasis than those with weak or negative stainings (87.5% vs 34.5%; P = 0.014). Only 25% of the benign specimens had weak or negative staining for CXCR4. Functioning CXCR4 receptor was expressed on HeLa cells. SDF-1alpha provoked significant signal transduction events, including chemotaxis and rescue from apoptosis. These actions were apparently mediated by the activation and phosphorylation of the extracellular signal-regulated kinase 1/2 and AKT pathways. We conclude CXCR4 expression is associated with cervical adenocarcinoma cell migration and proliferation, and primary cervical adenocarcinoma cells expressing CXCR4 are significantly more likely to metastasize to pelvic lymph nodes.