Purpose <p>Carbonic anhydrase IX (CA-IX) is a hypoxia-regulated enzyme involved in pH homeostasis and tumor progression. This study aimed to investigate the effects of acetazolamide (AAZ), a CA-IX inhibitor, on mTOR signaling components in CAKI-2 renal carcinoma cells.</p> Methods <p>CAKI-2 cells were treated with AAZ and rapamycin (RAPA). Cell viability was assessed using the WST-1 assay. The expression levels of CA-IX, mTOR, phosphorylated mTOR (p-mTOR), 4E-BP1, and phosphorylated 4E-BP1 (p-4E-BP1) were evaluated using ELISA, Western blot, and immunofluorescence staining.</p> Results <p>AAZ treatment significantly reduced CA-IX levels (<i>p</i> &lt; 0.0001), while no significant changes were observed in mTOR, p-mTOR, 4E-BP1, or p-4E-BP1 protein levels. In contrast, RAPA treatment significantly decreased the levels of p-mTOR and p-4E-BP1 (<i>p</i> &lt; 0.05). Immunofluorescence analysis confirmed the downregulation of CA-IX following AAZ exposure, with no notable changes in the localization or intensity of mTOR and 4E-BP1 signals.</p> Conclusion <p>Although AAZ effectively inhibits CA-IX expression, it does not significantly impact mTOR pathway activation in CAKI-2 cells under normoxic conditions. Further studies are warranted to evaluate the role of CA-IX inhibition under hypoxic or combinatorial treatment settings.</p>