Objectives <p>To explore the effect of <i>Pollen Pini</i> (PP) on SMMC-7721 hepatoma cells.</p> Methods <p>The anti-proliferative effects of PP on SMMC-7721 cells were evaluated using cell counting kit-8 assay following 48 h treatment with concentrations ranging from 1.25 to 37.50 µg/µL Flow cytometry analysis at the half maximal inhibitory concentration (IC₅₀) revealed significant apoptosis induction and cell cycle alterations. For <i>in vivo</i> evaluation, SMMC-7721 xenograft-bearing nude mice were administered either vehicle (0.9% NaCl) or PP (500 mg/kg) via intraperitoneal injection every other day for 3 weeks. Subsequent tumor analysis included mass spectrometry-based proteomics and examination of phosphoinositide-3 kinase (PI3K)/protein kinase B (Akt)/Myc pathway proteins by immunohistochemistry and Western blot. Combination therapy with 25 µmol/L PI3K inhibitor and PP (IC₅₀) showed optimal efficacy, with Western blot revealing maximal protein modulation at 24 h.</p> Results <p>PP had an anti-tumor effect on SMMC-7721 cells <i>in vitro</i>, with the IC₅₀ concentration of 18.94 mg/mL. PP could promote the death of SMMC-7721 cells (<i>P</i>&lt;0.01) and regulate the cell cycle more in G₀/G₁ phase cells (<i>P</i>&lt;0.01). After the treatment effect of PP, the protein content of SMMC-7721 cells changed and the contents of cancer-promoting proteins PI3K, Akt and Myc decreased (P&lt;0.01). PI3K inhibitor could reduce the proliferation of SMMC-7721 cells, and PI3K inhibitor combined with PP significantly reduced the expression of PI3K in SMMC-7721 cells (<i>P</i>&lt;0.01).</p> Conclusion <p>PP has an anti-tumor effect on SMMC-7721 cells through the PI3K-Akt signaling pathway.</p>