Purpose: To investigate the associations between programmed cell death ligand-1 (PD-L1) on tumor cells (TCs) or PD-L1 on tumor-infiltrating immune cells (TIICs) and the microsatellite instability (MSI) status in colorectal cancer (CRC). Methods: In total, 238 CRC patients were enrolled. PD-L1 expression and MSI status were studied by immunohistochemical staining and polymerase chain reaction. The χ2 test was used to compare characteristics. The Kaplan–Meier method was used for survival analysis. Cox proportional hazards models were used to determine the prognostic influence of clinicopathological factors. Results: Eighteen patients (7.6%) were had MSI-high (MSI-H) CRC. The number of patients with PD-L1 expression on TCs, stromal TIICs and intraepithelial TIICs was 13 (5.5%), 64 (26.9%) and 45 (18.9%), respectively. The MSI-H phenotype was significantly associated with younger age, right sidedness, mucinous component, high grade, stromal TIICs expressing PD-L1 (P = 0.042) and intraepithelial TIICs expressing PD-L1 (P < 0.001), but not TCs expressing PD-L1. PD-L1-expressing TCs were an independent marker of poor prognosis [hazard ratio (HR) = 3.387, P = 0.003], and PD-L1-expressing stromal TIICs were an independent marker of good prognosis (HR = 0.551, P < 0.001). Conclusions: PD-L1-expressing TCs were a marker of poor prognosis; in contrast, PD-L1-expressing TIICs were a marker of good prognosis. The MSI-H phenotype was associated with the presence of PD-L1-expressing TIICs, but not of PD-L1-expressing TCs.