Substantial studies have demonstrated that the initiation and progression of cervical cancer were closely associated with human papillomavirus (HPV) E6 and E7 oncogenes. The therapeutic strategy with ribozyme or antisense oligonucleotides to inhibit the expression of HPV E6 or E7 oncogenes showed effect to some degree, but problems such as low efficiency, short-period maintenance, and high cost still remain. The aim of this study was to investigate in vitro and in vivo the effect of HPV 16 E6 small interfering RNA (HPV 16 E6 siRNA) on cervical cancer cell line CaSki cells. The specific siRNA of HPV 16 E6 was synthesized and transfected into CaSki cells by liposome. The number of apoptotic cells, HPV 16 E6 messenger RNA (mRNA) level, and E6 protein expression were measured before and after the transfection by flow cytometry, reverse transcriptase-polymerase chain reaction, and Western blot, respectively. Cervical cancer in nude mice was established, and siRNA was injected directly into the nude mice peritoneal cavity or subcutaneous tumor. The efficiency of siRNA was evaluated by tumor volume change, HPV 16 E6 protein expression, and apoptosis of tumor cells. Apoptosis rate of CaSki cells at days 1, 2, 5, and 9 after siRNA transfection were 7.7%, 11.8%, 37.4%, and 12.6%, respectively. The mRNA level of HPV 16 E6 at the same time points were reduced by 77%, 83%, 59%, and 41%, respectively. But the mRNA level of beta-actin, as an internal control, showed no significant change. The inhibition rates of E6 protein synthesis at days 1, 2, 5, and 9 after the transfection were 79.7%, 80.4%, 71.3%, and 57.4%, respectively, whereas the protein levels of Lamin A/C, as internal control, had no change. In vivo, E6 siRNA administration groups showed a dramatic effect in inhibiting tumor growth, suppressing expression of E6 protein, and inducing tumor necrosis and apoptosis as compared with the control group. Direct injection of siRNA into subcutaneous tumor resulted in tumor suppression effect similar to that via the peritoneal cavity, and with additional injection better results could be achieved in cervical cancer CaSki cells. RNA interference exists, and the interference to HPV 16 E6 is specific and highly efficient both in vitro and in vivo.