Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. In this study, we examined the role of TP expression in relation to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced cytotoxicity in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1650. Treatment with 17-AAG (0.1–1μM) resulted in a decrease in cellular TP protein and mRNA levels, which was accompanied by a downregulation of phosphorylated MKK1/2-ERK1/2 and AKT protein levels. The 17-AAG treatment disrupted the interaction between HSP90 and TP and triggered TP protein degradation through the ubiquitin-26S proteasome pathway. Specific inhibition of TP expression by siRNA further enhanced the cell death and growth inhibition that had been induced by 17-AAG. An enhancement of ERK1/2 or AKT activation by transfecting the cancer cells with constitutively active MKK1/2 or AKT expression vectors significantly restored the 17-AAG-reduced TP protein levels as well as cell viability. In contrast, a combination of U0126 (MKK1/2 inhibitors) or LY294002 (PI3K inhibitor) further decreased the TP expression and cell viability induced by 17-AAG. Moreover, 17-AAG enhanced the cisplatin-induced cytotoxic effect through downregulation of the cisplatin-induced TP expression and ERK1/2 and AKT activation. Taken together, our results suggest that the down-modulation of TP protein induced by 17-AAG represents a key factor in enhancing the cytotoxic effects of cisplatin in NSCLC cells.