AIM: To explore the mechanism of action of the microtubular inhibitor of CA-4 derivative LGD5 on human cervical cancer HeLa cells. METHODS: HeLa cells were selected and divided into blank group, CA-4 positive control group, and different concentrations of LGD5 were formed into the experimental group. MTT was used to investigate the growth inhibition of LGD5 on HeLa cells and to determine the assay concentration. Cell morphology and apoptosis were observed before and after drug administration by inverted microscope and acridine orange staining.  Immunofluorescence staining was used to examine the effect of LGD5 on microtubules using DAPI. The effect of LGD5 on cell cycle by PI flow cytometry. Protein immunoblotting was used to examine the effect of LGD5 on cyclins and apoptosis-related proteins. RESULTS: MTT experiments showed that LGD5 inhibited HeLa cells in a time-and dose-dependent manner. Timed photography and acridine orange staining observed that LGD5 induced apoptosis in HeLa cells and produced significant chromatin agglutination and apoptotic bodies. Inhibition of microtubule polymerization in HeLa cells by LGD5 was observed by DAPI staining. The PI flow cytometry results showed that LGD5 induced G2/M cycle arrest in HeLa cells, was time-dependent and dose-dependent within 12 h, and had a significant difference (P<0.01), apoptosis was induced after 24 h and it was time-dependent. The results of Western blot show that, LGD5 downregulates Cdc 2 and Cdc25C, upregulation of p-Cdc 2, and Cyclin B1 and p-histone H3, further verified that LGD5 induced G2/M cycle arrest in HeLa cells, besides, LGD5 caused increased Caspase 3 expression in HeLa cells, upregulated Caspase 9 and Bax, down-regulation of Pro-caspase 9 and Bcl-2, this result indicates that HeLa cell apoptosis induced by LGD5 is related to the mitochondrial pathway. CONCLUSION: The CA-4 derivative LGD5 inhibited microtubule polymerization in HeLa cells, induced their G2/M cycle arrest, and subsequently induced cell apoptosis through the mitochondrial pathway.