Objective To investigate the effect of miR-758-3p targeting and regulating COL4A1 on the malignant biological behaviors of hepatocellular carcinoma (HCC) cells. Methods From November 2023 to November 2024, the tissue and adjacent tissues of 33 HCC patients who underwent surgery in our hospital were subjected to immunohistochemical detection of the positive expression of COL4A1. qRT-PCR was used to detect the expression of miR-758-3p and COL4A1 mRNA in the tissues and HCC cell lines (HepG2, Hep3B, and Huh-7), as well as in the normal human liver cell line (LO2). Logarithmic growth phase HepG2 cells were divided into blank group, miR-NC group, miR-758-3p overexpression group, si-NC group, interference with COL4A1 group, miR-758-3p overexpression + empty vector group, and miR-758-3p overexpression + COL4A1 overexpression group. qRT-PCR was used to detect the expression of miR-758-3p and COL4A1 mRNA in the cells. The targeting relationship of miR-758-3p and COL4A1 was verified by dual luciferase assay. Cell proliferation, apoptosis, invasion and migration were detected by CCK-8 method and EdU staining, flow cytometry, Transwell assay and scratch assay. COL4A1, cyclin D1 (CyclinD1), apoptosis protein (Bax), matrix metalloproteinase (MMP)-9 and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway-related proteins were detected by Western blot. Nude mouse tumor formation experiment was used to detect the changes in tumor weight in vivo after transfection of cells and the expression of COL4A1, CyclinD1, Bax, MMP-9 and proteins of PI3K/AKT pathway. Results The expression of miR-758-3p decreased in HCC tissues and cells, while the mRNA and positive expression of COL4A1 increased (P＜0.05). The OD 450 EdU positivity rate, invasion number, healing rate, CyclinD1, MMP-9, p-PI3K/PI3K, p-AKT/AKT, COL4A1 mRNA and protein expression in the miR-758-3p overexpression group were lower than those in the miR NC group and blank group, while the apoptosis rate, miR-758-3p expression, and Bax expression were higher (P＜0.05). The OD 450, EdU positivity rate, invasion number, healing rate, CyclinD1, MMP-9, p-PI3K/PI3K, p-AKT/AKT, COL4A1 mRNA and protein expression in the COL4A1 interference group were lower than those in the si NC group, while the apoptosis rate and Bax expression were higher (P＜0.05). Overexpression of COL4A1 reversed the inhibitory effect of miR-758-3p overexpression on the malignant development of HCC (P＜0.05). miR-758-3p targeted and regulated COL4A1 (P＜0.05). The in vivo transplantation tumor experiment showed that overexpression of miR-758-3p could inhibit tumor growth, and overexpression of COL4A1 reversed the inhibitory effect of overexpression of miR-758-3p on tumor growth.Conclusion MiR-758-3p inhibits the malignant biological behavior development of HCC by targeting COL4A1