Abstract: To examine the regulatory mechanisms of oligopeptides derived from soft-shelled turtle protein on tubulin activity in tumor cells, this study employed three specific oligopeptides D-7-A, E-7-W, and G-8-V obtained from soft-shelled turtle protein. The objective was to assess their impact on tubulin polymerization and depolymerization, investigate their interaction with tubulin, and elucidate the mechanisms induce apoptosis in tumor cells. The results showed that the three oligopeptides exhibited similarities to vincristine and acted as inhibitors of tubulin polymerization, their respective IC₅₀ values was 3.72, 5.01, and 5.95 µmol/L. Furthermore, the molecular docking analysis revealed that D-7-A interacted with the active center of α-tubulin 4X1I, specifically binding to Ser178 and Thr179. The active center of E-7-W was found to be bound by Gln15, Thr225, Tyr224, Tyr210 and Arg214. Similarly, G-8-V was observed to bind to Gln11, Ser178 and Asp329 at the active center, exerting a significant impact on α-tubulin. The results of cell experiments showed that D-7-A, E-7-W and G-8-V had dose-dependent inhibitory effects on A549 cells, with IC₅₀ values of 2003±72, 1877±102 and 1789±137 μmol/L, respectively. The G-8-V exhibited the most potent inhibitory effect on A549 cells, leading to the induction of apoptosis. Moreover, the apoptosis rate demonstrated a significant increase with prolonged drug treatment time. The underlying mechanism of action involved the inhibition of tubulin polymerization and the induction of apoptosis by perturbing the valence bond of α-tubulin.