Background: Oral uracil and tegafur (UFT)/leucovorin (LV) are widely used as a standard adjuvant chemotherapy for colorectal cancer (CRC). On the other hand, immune checkpoint inhibitors such as pembrolizumab are being developed for the treatment of patients with CRC with high microsatellite instability or a mismatch repair deficiency. However, the effects of standard chemotherapy on the expression levels of immunotherapy targets are unknown. In the present study, we examined the gene expression levels of immunotherapy targets in tumor tissues before and after UFT/LV chemotherapy in patients with CRC. Methods: The subjects were 80 patients with CRC who were scheduled to undergo surgery. UFT (300 mg/m2/day) and LV (75 mg/day) were administered for 2 weeks before surgery. Using an RT-PCR assay, the gene expression levels of four immunotherapy targets, CD274, CTLA4, IDO1 and LAG3, were quantitatively evaluated in paired samples of tumor-biopsy specimens obtained before chemotherapy (pre-samples) and resected-tumor specimens obtained after chemotherapy (post-samples). Results: In the pre-samples, no differences in the gene expression levels of the four immunotherapy targets were observed between the right- and left-sided colorectal cancer biopsy specimens, but the gene expression levels of LAG3 were significantly higher in elderly patients (≥75 years) than in younger patients (<75 years) (P = 0.0027). When the pre- and post-samples were compared, the gene expression levels of CTLA4 and LAG3 were significantly higher after UFT/LV chemotherapy (P = 0.0003 and P = 0.0111, respectively). Interestingly, these increases in gene expression were only observed in left-sided tumors (P < 0.0001 and P = 0.0056, respectively), and not in right-sided tumors (P = 0.3922 and P = 0.4085, respectively). Conclusions: The increases in the gene expressions of the immunotherapy targets CTLA4 and LAG3 after oral chemotherapy with UFT/LV were specific to left-sided colorectal tumors, suggesting that immunotherapy strategies for patients with CRC after standard chemotherapy should be considered separately for right- and left-sided colorectal tumors. Funding: None Disclosure: All authors have declared no conflicts of interest.