Abstract: U-104, an effective inhibitor of carbonic anhydrases (CAs), has been shown as a potential anti-tumor drug in several human cancer types.However, the downstream mechanisms of U-104 and its functions in tongue squamous cell carcinoma (TSCC) remain unclear. It is neither confirmed that whether the anti-tumor effects of U-104 are dependent on CA9 and CA12. In this work, we found differentially expressed genes (DEGs) and potential cellular processes regulated by U-104 through RNA sequencing. The cell death-related, cell proliferation, migration and response to drug cellular processes were among the top GO (gene ontology) processes, which were consistent with the observed biological effects upon U-104 treatment in TSCC15 cells. Furthermore, knockdown (KD) of CA9 or CA12 completely eliminated the U-104 effects on the cell migration, cell death, and the expression of critical DEGs. All together, our study suggests the regulatory mechanisms of U-104 at the transcriptome level and demonstrates the anti-tumor functions of U-104 dependent on CA9 and CA12 in TSCC. Our findings expand the current knowledge on the anti-tumor functions of U-104 and provide a potential therapeutic option for TSCC.