Hiwi, also named PiwiL1, is a human homologue of the Piwi family which is associated with stem cells and is overexpressed in several types of cancers. In the present study, we aimed to investigate the role of Hiwi in cervical carcinogenesis. Immunochemical analysis showed a significantly higher frequency of Hiwi staining in high-grade squamous intraepithelial lesions (HSILs) and cervical cancer tissues when comparing with the frequency in normal cervices. Particularly, Hiwi staining was restricted to basal cells of the normal cervix and was associated with the progression of cervical cancer and chemotherapy resistance. We further found that ectopic Hiwi increased the chemical resistance in SiHa cells, and silencing of Hiwi in HeLa cells decreased the cell viability. In addition, as a cancer stem cell marker, Hiwi promoted the tumorsphere formation in vitro and tumorigenicity in vivo and elevated the expression of several stem cell self-renewal-associated transcription factors, in spite of inhibited the proliferation. These results suggest that Hiwi may participate in the carcinogenesis of cervical cancer and may be a potential therapeutic target molecule for cervical cancers.