Tobacco smoking is associated with an increased risk of oral leukoplakia and head and neck cancer. Although it has recently been reported that the establishment of an immunosuppressive microenvironment in oral potentially malignant disorders may lead to malignant transformation, it is unclear whether the microenvironments of oral potentially malignant disorders differ according to smoking status. We examined differences in programmed death-ligand 1 (PD-L1) expression and subepithelial CD163+ TAM and CD8+ cell/lymphocyte counts in the microenvironment of oral leukoplakia of smoking and non-smoking patients and investigated their associations with malignant transformation. Pathology reports and original biopsy request forms from 1995-2015 were retrospectively reviewed. Lesions clinically characterized as white plaques/lesions of the oral mucosa and pathologically diagnosed as oral epithelial dysplasia were included. Immunohistochemistry was performed to evaluate PD-L1 expression and subepithelial CD163+/CD8+ cell counts. The significance of prognostic factors in predicting malignant transformation was determined using Cox regression analysis. Statistical significance was defined as P<0.05. In total, 200 patients with oral leukoplakia were selected. The mean age at diagnosis was higher in non-smoking patients (n = 141; 66.9 years) than in smoking patients (n = 59; 60.5 years). The 5-year cumulative malignant transformation rate was higher in non-smoking patients than in smoking patients (9.3% vs. 3.0%, respectively). Oral leukoplakia was associated with significantly higher PD-L1 expression and increased numbers of subepithelial CD163+ cells in the non-smoking group compared with the smoking group. Non-smoking-related oral leukoplakia with positive PD-L1 expression was associated with a 6.97-fold (95% confidence interval: 2.14-22.7) increased risk of malignant transformation. The microenvironment of oral leukoplakia differed according to smoking status. A combination of smoking status and PD-L1 expression may predict malignant transformation in oral leukoplakia patients. This study highlights the importance of understanding the interaction between smoking and the microenvironment in oral leukoplakia.