Gastroenteric cancer is one of the most prevalent cancers and is responsible for most cancer-related deaths worldwide. Paclitaxel (PTX), a classical microtubule inhibitor, is indicated in the treatment of gastric/gastroenteric cancers. In the present study, trimethyl chitosan (TMC)-loaded PTX (TMC-PTX) was prepared and evaluated for its therapeutic effect in gastric cancers. A spherical shaped nanosized TMC-PTX particle was formed with high loading capacity (~30%) for PTX. The nanoparticles (NPs) showed a sustained release pattern (~70%) for up to 96 h of study period. The positively charged NPs were preferentially internalized by Caco-2 cells. TMC-PTX inhibited the gastric cell proliferation with an IC50 value of 0.6 µg in NCI-N87 cells while it was 1.26 µg in the SGC-7901 cell line after 24 h exposure. The apoptosis assay (Annexin V/PI) showed a large presence of cells in the early and late apoptosis chamber, while cell cycle analysis showed a marked G2/M phase arrest (50-60%) in NCI-N87 and SGC-7901 cell lines indicating its potent anti-proliferative effect. The in vivo antitumor study in NCI-N87 and SGC-7901 bearing xenograft model showed a superior chemotherapeutic efficacy for TMC-PTX NP. The NP group significantly reduced the tumor growth with no obvious sign of systemic side-effects (safety). Collectively, our results suggest that the microtubule inhibitory effect of PTX-loaded polymer NP could be a promising system for the treatment of gastroenteric cancers.