Ergosterone has been proved to have potential antitumor effect on H22 tumor-bearing mice, but the antitumor mechanism and key regulators are still unclear. The current study was aimed to explore the key regulators responsible for antitumor of ergosterone using whole transcriptome and proteome analysis in H22 tumor-bearing mice model. The model of H22 tumor-bearing mice was constructed according to the histopathological data and biochemical parameters. The isolated tumor tissues of different treatment groups were subjected to transcriptomic and proteomic analysis. Our findings demonstrated that 472 differentially expressed genes and 658 proteins were identified in the tumor tissue of different treatment groups through RNA-Seq and liquid chromatography with tandem mass spectrometry-based proteomic analysis, respectively. The combined omics analysis revealed three critical genes/proteins, including Lars2, Sirpα and Hcls1 that could play a role in antitumor pathways. Furthermore, Lars2, Sirpα and Hcls1 genes/proteins, as key regulators of the antitumor effect of ergosterone, were verified by qRT-PCR and western blotting methods, respectively. In summary, our study provides new insights into analysing the antitumor mechanism of ergosterone from the point of view of gene and protein expression and will encourage further development of the antitumor pharmaceutical industry.