Background and Aims Hepatocellular carcinoma (HCC) is one of the most common malignancies, with high incidence and mortality. The JAK/STAT signaling pathway plays a crucial role in regulating cell proliferation, apoptosis, and immune responses, and its persistent activation is closely associated with the development of HCC. This study aimed to investigate the effects of the JAK2 inhibitor AG490 and the STAT3-related inhibitor rapamycin (RPM) on the biological behaviors of HCC cells and their underlying molecular mechanisms.Methods Human hepatoma HepG2 cells were divided into four groups: blank control, AG490, RPM, and AG490+RPM. Cell proliferation, migration, and apoptosis were assessed by MTT assay, scratch test, and flow cytometry, respectively. ELISA and qRT-PCR were used to detect the protein and mRNA expression of Bax, Bcl-2, caspase-3, survivin, and c-Myc. A Western blot analysis was performed to examine the expression and phosphorylation levels of JAK2 and STAT3.Results Compared with the blank control group, the AG490, RPM, and AG490+RPM groups showed significantly decreased cell proliferation and migration abilities, as well as increased apoptosis, with the most pronounced effects observed in the AG490+RPM group (all P<0.05). Inhibitor-treated groups showed elevated expression of Bax and caspase-3, decreased expression of Bcl-2, survivin, and c-Myc, with the most significant changes in the AG490+RPM group (all P<0.05). In addition, the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 were significantly reduced in all treated groups, with the lowest levels in the AG490+RPM group (all P<0.05).Conclusion Inhibitors targeting the JAK/STAT pathway significantly suppress proliferation and migration, and induce apoptosis in HCC cells, possibly by downregulating p-JAK2 and p-STAT3, as well as modulating genes related to apoptosis and proliferation. The combined use of AG490 and RPM exhibits superior antitumor effects, suggesting that multi-target blockade of the JAK/STAT pathway may represent a promising therapeutic strategy for HCC.