Andrographolide (Androg) has been reported to contain antiviral and antitumor activities, but the effects of Androg on human papillomavirus (HPV) infection and cervical cancer have not been elucidated. This study investigated the effects of Androg and its derivatives, namely, 14-deoxy-11,12-didehydroandrographolide (14-DDA) and 3,19-isopropylidene andrographolide (IPAD), on HPV16 pseudovirus (HPV16PsV) infectivity, HPV16 E6 oncogene expression and cervical cancer cell apoptosis. The result demonstrated that all compounds inhibited HPV16PsV infection and that 14-DDA showed the highest potency. Only Androg suppressed long control region (LCR) transcription activity of HPV16 in transiently transfected C33A cells and significantly inhibited E6 oncogene expression in SiHa cells in a dose-dependent manner. A twofold subcytotoxic concentration of IPAD exhibited an inhibitory effect on E6 oncogene expression at 48-h posttreatment. Interestingly, p53 protein was restored in a downstream process and was detected earlier by IPAD treatment than by Androg treatment. This result corresponded to the level of cell apoptosis and cell cycle arrest at the G2/M phase. E6 oncogene expression was also suppressed in CaSki cells treated with Androg and IPAD leading to cell apoptosis. These findings imply that Androg and its derivatives have different activities and may be effective agents for HPV prevention and cervical cancer treatment.