The aim of the present study was to investigate the role of chemokine (C-X-C motif) ligand 12 (CXCL12) and its receptor, chemokine (C-X-C motif) receptor 4 (CXCR4) in the pathogenesis of adenomyosis (AD). Immunohistochemistry and reverse transcription-quantitative polymerase chain reaction analysis were used to measure the protein and mRNA expression of CXCL12 and CXCR4 in eutopic endometrial and ectopic foci tissue samples. Samples from a total of 36 patients with AD (study group) were compared with endometrial tissue samples from 33 patients who underwent uterine fibroids surgery (control group) during the same period. All data are presented as the mean +/- standard deviation and were analyzed with SPSS software (version 16.0). Analysis of variance was used for between group analysis and pairwise comparison was performed using Fisher's least significant difference post hoc test. The results of the present study revealed that CXCL12 and CXCR4 protein expression was significantly increased in ectopic foci tissue compared with eutopic endometrial tissue samples from patients with AD. CXCL12 and CXCR4 protein expression in ectopic foci and eutopic endometrial tissue samples were significantly increased compared with the control group (P< 0.05 for between group comparisons). No significant differences were identified in CXCL12 and CXCR4 protein expression between the proliferative and secretory phases within each group. Furthermore, CXCL12 and CXCR4 mRNA expression was significantly increased in ectopic foci tissue and eutopic endometrial tissue compared with the control group (P< 0.05 for between group comparisons). CXCL12 mRNA expression was markedly increased in ectopic foci tissue compared with eutopic endometrial tissue of patients with AD. The expression of CXCR4 mRNA was significantly increased in eutopic endometrial tissue compared with ectopic foci tissue and the control group (P< 0.05 for between group comparisons). No significant differences were identified in CXCL12 and CXCR4 mRNA expression between proliferative and secretory phase within each group. In conclusion, CXCL12 and CXCR4 may induce the ectopia, and promote the spread and localized growth of endometrial cells in the development of AD.