Taxol (paclitaxel) and vinblastine (VBL) are both efficacious chemotherapeutic agents that target the microtubules of tumor cells, but each functions in a mutual antagonistic manner. Op18/stathmin is a small molecular phosphoprotein which promotes depolymerization of microtubules. Non-small cell lung cancer (NSCLC) NCI-H1299 cells were employed to compare the curative effects of VBL and Taxol and explore the correlation between drug sensitivity and Op18/stathmin signaling. The present study found that VBL obviously promoted cellular apoptosis and initiated activation of caspase 3 and 9, and inhibited cell proliferation and colony formation, as well as cell migration in the NCI-H1299 cells in contrast with Taxol. VBL did not affect the expression of Op18/stathmin, but increased its phosphorylation at all 4 serine sites. Conversely, Taxol mainly decreased the expression of Op18/stathmin and the phosphorylation at Ser25 and Ser63 sites. Silencing of Op18/stathmin by RNA interference (RNAi) led to a great reduction in the differences in the cell proliferation inhibition between VBL and Taxol. VBL treatment notably weakened the expression of PP2A, Bcl-2, NF-κB and interleukin-10 (IL-10) and autocrine IL-10 compared with Taxol; whereas PP2A was substantially increased following Taxol induction. High expression of Op18/stathmin was found to be negatively correlated with the sensitivity of Taxol in the NSCLC cells, but had a minor impact on VBL cytotoxicity. These findings revealed that both VBL and Taxol induce cell apoptosis through Op18/stathmin, but the mechanisms are completely different. VBL is an attractive alternative to the treatment of Taxol-resistant tumors with high expression of Op18/stathmin.