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Progestin usage can alter the histologic and cytologic features of HSIL, which may potentially lead to the under-diagnosis of this precancerous lesion. The objective of the present study was to assess the relationship between the usage of progesterone-based contraceptives and the cytologic features of high-grade squamous intraepithelial lesion (HSIL) of the cervix. In this case-control study, we assembled 46 cases of cervical HSIL (CIN 3), including samples from 26 patients with known history of progestin usage (study group) and 20 samples from patients with no history of any exogenous hormone usage (control group). Cell image analysis was performed on all samples using a proprietary software. Immunohistochemical studies for Ki67, p16, estrogen receptor and progesterone receptor were performed on all cases, as was RNA in situ hybridization for HPV subtypes 16 and 18. Compared with the control group cases, the average nuclear size (21.5±1.80 μm) and the nuclear: cytoplasmic ratio (0.28±0.015) of HSILs in the study group cases were significantly smaller (P=0.026) and reduced (P=0.005), respectively. In addition, the study group cases showed reduced nuclear atypia and pleomorphism and significantly reduced mitotic figures (1.74±1.86/mm in study group versus 5.94±1.3/mm in control group, P<0.0001). The later was likely a consequence of the significantly reduced mitotic figures in the superficial and middle epithelial layers of the study group cases compared with their control group counterparts (0.73±0.88/mm vs. 7.3±2.6/mm, P<0.0001). Progestin usage has no discernible effects on p16 immunoreactivity, Ki67 proliferative index, hormone receptor expression, and HPV RNA levels of HSIL lesions. Being aware of progestin induced morphologic changes on those cervical precancerous lesions will be ultimately improve patient care quality in our daily practice.