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<div><p>Objective</p><p>This study aimed to investigate the influence of pine pollen (PP) on hepatocellular carcinoma (HCC) behavior <i>in vitro</i> and <i>in vivo</i> and explore its mechanism of action by focusing on the phosphatidylinositol 3-kinase/protein serine-threonine kinase (PI3K/AKT) signaling pathway and α-Enolase (<i>ENO1</i>) gene expression.</p><p>Methods</p><p>We performed a bioinformatics analysis of <i>ENO1</i>. HCC cells overexpressing <i>ENO1</i> were developed by lentivirus transfection. Cell proliferation, invasion, and migration were assessed using the cell cytotoxicity kit-8 assay, transwell assay, cell scratch test, and <i>ENO1</i> inhibiting proliferation experiment. Protein expression was analyzed using Western blot. The <i>in vivo</i> effects of PP on HCC xenografts were also assessed in mice. The serum of nude mice in each group was analyzed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and AST/ALT. The tumor blocks of nude mice were weighed, and proteins were extracted for Western blot.</p><p>Results</p><p>Compared to normal cells, the phosphorylation of <i>ENO1</i> at the S27 site was most significant in HCC cells and was closely related to cell proliferation. <i>In vitro</i>, the PP solution inhibited the proliferation, invasion, and migration of <i>ENO1</i> overexpressing cells compared with empty-vector-transfected cells. In mice bearing HCC, PP injection inhibited the overexpression of <i>ENO1</i>, affected serum ALT, AST, and AST/ALT levels, and reduced tumor weight. However, the expression of proliferation-related proteins in tumors overexpressing <i>ENO1</i> was higher than in empty transfected tumors.</p><p>Conclusion</p><p>PP inhibits HCC by regulating the expression of <i>ENO1</i> and <i>MBP-1</i> and suppressing the PI3K/AKT pathway by inhibiting C-MYC and erb-B2 receptor tyrosine kinase 2.</p></div>