The ansamycin-based HSP90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin) combats tumors and has been shown to modulate cellular sensitivity to radiation, prompting researchers to use 17-AAG as a radiosensitizer. 17-AAG causes the degradation of several oncogenic and signaling proteins. We previously demonstrated that oxidative stress activates serine/threonine kinase 38 (STK38), a member of the protein kinase A (PKA)/PKG/PKC-like family. In the present study, we investigated how 17-AAG affects STK38 expression, and evaluated STK38's role in the regulation of radiosensitivity. We found that 17-AAG depleted cellular STK38 and reduced STK38's kinase activity. Importantly, 17-AAG downregulated the stk38 gene expression. Deletion analysis and site-directed mutagenesis experiments demonstrated that Sp1 was required for the stk38 promoter activity. Treatment with 17-AAG inhibited Sp1's binding to the stk38 promoter by decreasing the amount of Sp1 and knocking down Sp1 reduced STK38 expression. Moreover, 17-AAG treatment or STK38 knockdown enhanced the radiosensitivity of HeLa cells. Our data provide a novel mechanism, mediated by stk38 downregulation, by which 17-AAG radiosensitizes cells.