Objective To analyze the mechanism of loongmycin-induced cell cycle arrest in triple-negative breast cancer cell line by bioinformatics. Methods The triple-negative breast cancer cell line, MDA-MB-468, was treated with loongmycin for 48 and 72 h. The half inhibition concentration (IC₅₀) was calculated and the cell cycle was analyzed by flow cytometry. The MDA-MB-468 cells treated with 0.8 μmol/L loongmycin for 48 h and the control cells without drug treatment were selected for transcriptome sequencing. After the data were filtered by quality control, the differential gene expression was analyzed using DESeq2 1.16.1 software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional analyses and gene set enrichment analysis were performed. The protein-protein interaction network was analyzed by STRING and the docking prediction was performed using AutoDock 1.5.6 software. Results The IC₅₀ values of MDA-MB-468 cells intervened by loongmycin for 48 and 72 h were 2.733 and 0.866 μmol/L, respectively. The results of flow cytometry showed that loongmycin arrested MDA-MB-468 cell line in G₂/M phase after intervening for 48 h. A total of 1 764 differentially expressed genes were screened after intervention with loongmycin. The differentially expressed genes were mainly located in nuclear factor kappa B (NF-κB)-mediated tumor necrosis factor alpha (TNF-α) pathway and P53 pathway. G protein subunit gamma 7 (GNG7), G protein subunit gamma 11 (GNG11), C-X-C motif chemokine ligand 8 (CXCL8), adenylate cyclase 2 (ADCY2) and other genes may be the key nodes in the gene network of drug action. Loongmycin and DNA molecules, topoisomerase, gene expression regulator (proteasome 20S subunit beta 5 [PSMB5] and SET domain containing histone lysine methyltransferase 7 [SETD7]) had good combination and interaction functions. Conclusion Loongmycin has similar functions to rebeccamycin; it exerts anti-tumor activity by binding with DNA molecules in DNA-topoisomerase Ⅰ complex; and it can also affect the expression of GNG7, GNG11, CXCL8, ADCY2 and other genes by binding gene expression regulatory proteins PSMB5 and SETD7, and eventually up-regulate NF-κB-mediated TNF-α pathway and P53 pathway, leading to G₂/M arrest in breast cancer cells, which plays an anti-tumor role.