Crocetin is a carotenoid extracted from Gardenia jasminoides, one of the most popular traditional Chinese medicines, which has been used in the prevention and treatment of various diseases. The present study is aimed at clarifying the effect of crocetin on gene expression profiling of HepG2 cells by RNA-sequence assay and further investigating the molecular mechanism underlying the multiple biofunctions of crocetin based on bioinformatics analysis and molecular evidence. Among a total 23K differential genes identified, crocetin treatment upregulated the signals of 491 genes (2.14% of total gene probes) and downregulated the signals of 283 genes (1.24% of total gene probes) by ≥2-fold. The Gene Ontology analysis enriched these genes mainly on cell proliferation and apoptosis (BRD4 and DAXX); lipid formation (EHMT2); cell response to growth factor stimulation (CYP24A1 and GCNT2); and growth factor binding (ABCB1 and ABCG1), metabolism, and signal transduction processes. The KEGG pathway analysis revealed that crocetin has the potential to regulate transcriptional misregulation, ABC transporters, bile secretion, alcoholism, systemic lupus erythematosus (SLE), and other pathways, of which SLE was the most significantly disturbed pathway. The PPI network was constructed by using the STRING online protein interaction database and Cytoscape software, and 21 core proteins were obtained. RT-qPCR datasets serve as the solid evidence that verified the accuracy of transcriptome sequencing results with the same change trend. This study provides first-hand data for comprehensively understanding crocetin targeting on hepatic metabolism and its multiple biofunctions.