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BACKGROUND AND PURPOSE The zinc finger transcription factor Snail is aberrantly activated in many human cancers and strongly associated with poor prognosis. As a transcription factor, Snail has been traditionally considered an "undruggable" target. Here, we identified a potent small molecule inhibitor of Snail, namely trimethoprim, and investigated its potential antitumor effects and the underlying mechanisms. EXPERIMENTAL APPROACH The inhibitory action of trimethoprim on Snail protein and the related molecular mechanisms were revealed by molecular docking, biolayer interferometry, immunoblotting, immunoprecipitation, qRT-PCR, pull-down, and cycloheximide pulse-chase assays. The anti-proliferative and anti-metastatic effects of trimethoprim via targeting Snail were tested in multiple cell-based assays and animal models. KEY RESULTS This study identified trimethoprim, an antimicrobial drug, as a potent anti-tumor agent via targeting Snail. Molecular modeling analysis predicted that trimethoprim directly binds to the arginine-174 pocket of Snail protein. We further discovered that trimethoprim strongly interrupts the interaction of Snail with CREB-binding protein (CBP)/p300, which consequently suppresses Snail acetylation and promotes Snail degradation through ubiquitin-proteasome pathway. Furthermore, trimethoprim sufficiently inhibited the proliferation, epithelial-mesenchymal transition (EMT), and migration of cancer cells in vitro via specifically targeting Snail. More importantly, trimethoprim effectively reduced Snail-driven tumor growth and metastasis to vital organs such as lung, bone, and liver. CONCLUSIONS AND IMPLICATIONS These findings indicate, for the first time, that trimethoprim suppresses tumor growth and metastasis via targeting Snail. This study provides insights for a better understanding of the anticancer effects of trimethoprim and offers a potential anti-cancer therapeutic agent for clinical treatment.