Ganoderma lucidum spore oil (GLSO) is a well-known dietary supplement that is beneficial for immunoenhancement and shows good antitumor efficacy. However, the anti-tumor mechanism of GLSO remains unclear. Three tumor samples were randomly taken from model group mice and H-GLSO group mice, and the experimental operations were performed. The results revealed that 340 proteins were upregulated and 599 proteins were downregulated after treatment with GLSO . WikiPathway enrichment analysis of the differentially expressed proteins revealed that the lipoxygenase (LOX), cyclooxygenase (COX) and cytochrome P450 (P450) mediated eicosanoid metabolism pathways in tumor tissues were significantly inhibited after treatment with GLSO. Further statistical analysis of the differentially expressed proteins in the eicosanoid metabolism pathway was conducted to visualize the relevant results. The results showed that after intervention with GLSO, key proteins of the eicosanoid metabolism pathway, including the Gpx1, Lta4h, Ephx2, Alox15, Alox5, Ptgr1, Cyp4f3, Cyp4f14, Ggt5, Acox3, Ptgs1, Tbxas1, Pla2g4a, and Cyp2j5 were significantly downregulated in the tumor tissues of hepatoma H22-bearing mice treated with GLSO.