Abstract Purpose: We recently reported that indoleamine 2, 3-dioxygenase (IDO) activity is significantly correlated with more distant metastasis and worse survival. The present study examined whether radiotherapy (RT) dose fractionation correlates with IDO-mediated immune activity in patients with early-stage NSCLC. Methods: Patients with newly diagnosed stage I-II NSCLC treated with either conventionally fractionated 3-dimensional conformal radiotherapy (3DCRT) or stereotactic body radiotherapy (SBRT) were analyzed. Levels of two key molecules associated with the IDO immune checkpoint, serum kynurenine and the kynurenine:tryptophan ratio (K:T ratio), were measured at pre-RT, during-RT, and 3-month post-RT. The relationship between disease control outcomes [overall survival (OS), progression free survival, and local/regional/distant failure rates] and absolute levels of these markers, as well as dynamic changes in their levels during RT, was studied. Results: Fifty-six patients (SBRT = 28, 3DCRT = 28) with early-stage NSCLC were studied. In all patients, higher kynurenine post-RT was significantly associated with worse OS ([HR, 1.25; 95% confidence interval (CI), 1.01–1.55; P = 0.044). No statistically significant differences in absolute kynurenine levels or the K:T ratio were observed in patients treated with 3DCRT or SBRT at any of the three time points. However, the absolute kynurenine levels rose significantly more post-RT in the 3DCRT patients with a median increase 0.721 ng/mL, compared to that of SBRT patients (0.115 ng/mL); P = 0.022. Conclusions: This study validated that elevated IDO activity correlated with worse survival outcomes in patients with early-stage NSCLC treated with definitive RT. Hypofractionated SBRT may have less immunosuppressive effect than 3DCRT, as measured by IDO.