<p>Colon cancer (CC) ranks as the third most common cancer and the second leading cause of cancer-related death worldwide. Despite significant advances in treatment and early intervention, the precise mechanisms of action for many targeted therapies remain to be fully elucidated. This study aimed to investigate the potential off-target effects of the AURKA inhibitor Alisertib in colon cancer. Transcriptomic analysis revealed a striking divergence between AURKA silencing and Alisertib treatment: AURKA knockdown predominantly affected cell cycle-related pathways, whereas Alisertib significantly modulated immune-related pathways. This functional heterogeneity at the transcriptomic level suggests that Alisertib's mechanism of action is not solely dependent on AURKA inhibition. Through bioinformatics prediction and experimental validation, we further identified ZAP70 as a leading candidate mediator of Alisertib's effects. Crucially, a series of in vitro and in vivo experiments demonstrated that Alisertib's ability to suppress malignant phenotypes and tumor growth in colon cancer cells was maintained irrespective of AURKA overexpression, confirming its AURKA-independent activity. Collectively, our findings systematically reveal that Alisertib exerts significant AURKA-independent antitumor effects in colon cancer, likely mediated through alternative mechanisms such as the regulation of ZAP70 and associated immune pathways. This study provides a novel perspective on the pharmacological action of Alisertib and its clinical application.</p>