Signal transducer and activator of transcription-1 (STAT1) is an important factor in various cellular processes. The cancer stem cell (CSC) is considered as a tumor-initiating cell that drives the inner hierarchy in many cancers including epithelial ovarian cancer (EOC). Here, we explored for the first time the regulation of STAT1 on stemness properties in chemoresistant EOC cells. The paclitaxel (PTX)-resistant EOC cell line (OV3R-PTX) was derived from PTX-sensitive OVCAR-3 cells treated by the PTX regimen. A single cell clone OV3R-PTX-B4 was selected by fluorescence-activated cell sorting. PTX-resistant cells grew slowly in conventional 2D and 3D cultures, but tumor xenograft with PTX-resistant cells grew fast in nude mice. Interestingly, OV3R-PTX-B4 cells shared the characteristics of CSCs and stemness properties were found to be increased in the non-adherent spheroid culture system. The PTX-resistant cells had a high expression of CSC-related markers and low expression of STAT1 that had a high methylation level of CpG in its promoter region. Overexpressed STAT1 suppressed stemness properties, cell proliferation, and colony formation and favored the overall survival of patients with EOC. In summary, these data indicate a regulatory mechanism of STAT1 underlying drug resistance and provide a potential therapeutic application for EOC patients with PTX resistance.