Indoleamine 2,3-dioxygenase-1 (IDO) is an immune regulatory enzyme expressed by most human tumors. IDO levels in tumor cells correlate with increased metastasis and poor patient outcome and IDO is linked to tumor cell resistance to immunotherapy, radiation therapy, and chemotherapy. Knowledge of tumor cell-autonomous effects of IDO, independent of its well-known role in regulating and suppressing anti-tumor immune responses, is limited. Clonal populations of A549 human lung adenocarcinoma cells stably transfected with anti-IDO shRNA or scrambled control shRNA were used to study IDO effects on drug sensitivity and resistance. IFNγ was used to induce IDO in those cells. We show, for the first time, that IDO mediates human tumor cell resistance to the candidate anticancer drugs FK866 (an NAD+ inhibitor), methoxyamine (MX, a base excision repair [BER] inhibitor) and approved anticancer drugs pemetrexed (a folate anti-metabolite) and gemcitabine (a nucleoside analogue), and combined treatment with pemetrexed and MX, in the absence of immune cells. Concurrent knockdown of IDO and thymidylate synthase (TS, a key rate-limiting enzyme in DNA synthesis and repair) sensitizes human lung cancer cells to pemetrexed and 5FUdR to a greater degree than knockdown of either target alone. We conclude that BER in IDO-expressing A549 cells plays a major role in mediating resistance to a range of approved and candidate anticancer drugs. IDO inhibitors are undergoing clinical trials primarily to improve antitumor immune responses. We show that targeting IDO alone or in combination with TS is a potentially valuable therapeutic strategy for cancer treatment, independent of immune activity and in combination with conventional chemotherapy.