Collectively, these findings demonstrate that EVO inhibits CRC cell functions by downregulating ASS1 expression, and further knocking down ASS1 on this basis can more significantly reduce arginine synthesis and enhance the inhibition of cell proliferation and migration by exerting an additive effect with EVO-mediated suppression of the Wnt/β-catenin/c-MYC pathway along with a greater reduction in ASS1 levels. Here, ASS1 is linked to colorectal carcinoma.