The homeobox gene Six1 is overexpressed in multiple human tumors, playing a role in promoting tumorigenesis and metastasis. The present study was aimed to investigate the clinical implications of Six1 expression in cervical cancer. Six1 messenger RNA (mRNA) and protein expression was detected by reverse transcription (RT) polymerase chain reaction and Western blotting, respectively, in human cervical cancer cell lines CaSki, HeLa, C33A and 20 normal cervical specimens, 21 specimens of cervical intraepithelial neoplasias (CINs), and 54 specimens of cervical cancer tissue, and the clinical implications of Six1 gene expression was analyzed. There was Six1 mRNA and protein overexpression in cervical cancer cell lines CaSki, HeLa, and C33A. The Six1 expression level was higher in CaSki and HeLa cells than in C33A cells (P < 0.05). Six1 mRNA and protein expression increased from normal cervical epithelial tissues, to CINs, and then to cervical cancer tissue (normal cervical epithelial tissue vs CIN, P < 0.05; normal cervical epithelial tissue vs cervical cancer, and CIN vs cervical cancer, P < 0.01). The status of Six1 overexpression was correlated to clinical staging and lymph node metastasis of cervical cancer (P < 0.01) but not to pathological grading, tumor size, and age of the patient (P > 0.05). Six1 was overexpressed in cervical cancer cell lines and in cervical cancer tissues. Alteration of Six1 expression might contribute to the occurrence and development of cervical cancer.