Acne is the eighth most common disease worldwide. Disease burden of acne such as anxiety, reduced self‐esteem, and facial scarring lowers the life quality of acne patients. Isotretinoin is the most potent treatment for moderate‐severe acne. However, the adverse events of isotretinoin especially teratogenicity limit its use. This study aims at investigating the therapeutical mechanisms of isotretinoin using bioinformatics analysis. Differentially expressed genes (DEGs) were filtered from microarray datasets GSE10432, GSE10433, and GSE11792. Functional and pathway enrichment analyses of DEGs were performed. Protein–protein interaction (PPI) network and module analyses were also conducted based on DEGs. Using isotretinoin for 1 week, LCN2, PTGES, and GDF15 were upregulated and might mediate sebocytes apoptosis and thus decreased sebum production; CCL2 originated from activated TNF signaling pathway and S100A7 could be related with “acne‐flare”. While treating with isotretinoin for 8 weeks, key genes were downregulated, including HMGCS1, HMGCR, FDFT1, MVD, IDI1, and FDPS, which may be associated with decreased sebum synthesis; HMGCS1, HMGCR, and FDFT1 also probably associated with apoptosis of sebocytes. There were only two common genes including ACSBG1 and BCAT2 which worked in both 1 week and 8 weeks and could associate with decreased sebum synthesis and apoptosis of sebocytes, respectively. These results indicate potential therapeutics and side effect mechanisms of isotretinoin in the acne treatment and provide a research direction to further investigate the therapeutic mechanism of isotretinoin and thus develop retinoid‐like compounds with similar curative effect and without teratogenicity.