Abstract Introduction: Cervical cancer is a common genital tract cancer. Although abundant screening and vaccination are beneficial for reducing the disease burden, the molecular mechanism underlying tumorigenicity and aggressiveness are not fully understood. Human papillomavirus (HPV) E6 protein was found to be responsible in the development of cervical cancer. Membrane complement regulatory proteins (mCRP), such as CD46, CD55, CD59, control complement activation on the surface of tumour cells and suggested to be important in contributing tumorigenesis in various types of cancers. In our recent study, we have reported that HPV E6 protein enriches the expression of CD55 in cervical cancer cells, which would further promote radio-resistance and cancer aggressiveness. In the present study, we would characterize the functional role of other two mCRPs CD46 and CD59 in contributing cervical carcinogenesis. We would further examine if triple knockout of CD46, CD55, CD59 would have synergistic effect on blockage of tumorigenesis in cervical cancer. Material and method: Flow cytometry was performed to analyse the expression level of CD46, CD55 and CD59 in cervical cell lines C33A, C4-1, CaSki, SiHa, and a HPV18-E6-overexpressing stable clone (18E6-5, origin C33A cells). In order to investigate the effect on the blockage of those mCRPs, 18E6-5 clone with high CD46, CD55 and CD59 expression were chosen for in vitro functional study using and CRISPR/Cas9 knockout. Results and discussion: The percentage of CD46(+), CD55(+) and CD59(+) cells were high in C4-1, CaSki, SiHa and 18E6-5 lines. The C33A cells demonstrate a low CD55(+) subpopulation while high CD46(+) and CD59(+) subpopulations. We found that single knockout of CD46 or CD59 supressed cell proliferation, sphere-forming, anchorage-independent cell growth capability, and sensitized cells to radiation treatment. In addition, triple knockout of CD46, CD55, CD59 remarkably inhibited those effects comparing with the effects of single and double knockout. Conclusion: Silencing of CD46, CD55, CD59 expression abolished the tumorigenic, radio-resistant and aggressive effects in cervical cancer cells, with triple knockout displayed the highest blockage effects. Those mCRPs would be responsible for tumorigenicity, radio-resistance and cancer aggressiveness in cervical cancer. Citation Format: Xuetang Mo, Thomas Ho-Yin Leung, Michelle Kwan-Yee Siu, Hextan Yuen-Sheung Ngan. Elucidating the potential role of membrane complement regulatory proteins (CD46, CD55, CD59) in tumorigenesis of cervical cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3816.