Objective To explore the effect of hsa-microRNA-203-3p (miR-203) targeted-regulating Cullin2 (CUL2) on the biological characteristics of human papillomavirus 16 (HPV16) positive cervical cancer cells. Methods A total of 10 patients underwent cervical cancer screening from September 2018 to September 2019 in the Department of Obstetrics and Gynecology, the Second Hospital of Shanxi Medical University. HPV genotyping was performed as single HPV16 positive, and pathological examination showed cervical squamous cell carcinoma (SCC). Ten corresponding paracancer normal tissue samples were collected as control group. Real-time quantitative reverse transcription PCR (qRT-PCR) was performed to detect the expression of miR-203 in cervical squamous cell carcinoma and corresponding adjacent tissues, cervical cancer cell line (SiHa) and human immortal keratinocyte line (HaCaT) cells. GO and KEGG enrichment were applied to analyze the functions and pathways miR-203 involved. The regulatory relationship between miR-203 and CUL2 were verified via TargetScan website and dual luciferase reporter assay. The miR-203 mimics or inhibitor were transfected into SiHa cells to establish cell models of high and low expression of miR-203,and the expressions of CUL2 mRNA and protein were detected by qRT-PCR and Western blotting. The proliferation, migration and apoptosis of SiHa cells were assessed by CCK-8, scratch assay and Annexin V-APC/PI double staining, respectively. Results Results of qRT-PCR indicated that, compared with the corresponding adjacent tissues and HaCaT cells, the relative expression level of miR-203 decreased obviously in both cervical SCC tissues and SiHa cells (P<0.01). The results of GO and KEGG enrichment methods showed that miR-203 was widely involved in the ubiquitination process and the signaling pathways involved in the malignancies. TargetScan website and dual-luciferase reporter assay showed that the targeting regulatory relationship existed between miR-203 and CUL2 (P<0.01). qRT-PCR and Western blotting indicated that overexpression of miR-203 reduced the expressions of CUL2 mRNA and protein (P<0.05 or P<0.01); While low expression of miR-203 up-regulated the expressions of CUL2 mRNA and protein (P<0.01). CCK-8, scratch experiments and Annexin V-APC/PI double staining method confirmed that overexpression of miR-203 decreased proliferation rate and migration rate of SiHa cells (P<0.01), and elevated cell apoptosis rate (P<0.05). In contrast, low expression of miR-203 increased the proliferation rate and migration rate of SiHa cells (P<0.01), and reduced the apoptosis rate(P<0.01). Conclusion The miR-203 might suppress the biological characteristics of HPV16-positive cervical cancer cells SiHa by targeting CUL2, and it was expected to become a new candidate gene in diagnosis and treatment of cervical cancer.