Background: Peripheral regulatory T cells (pTregs) might involve in tumor immune microenvironment. We aim to evaluate the correlation between pTregs and tumor immune microenvironment. Methods: pTregs status was determined from assessment of the pTreg/total T cell ratio (ratio of Foxp3 Treg-specific demethylated region (TSDR) to CD3G/CD3D demethylation) through epigenetic pattern of bisulfite pyrosequencing in long-term stored peripheral blood of 442 gastric cancer patients who received curative surgery. Immunohistochemical staining of multiple immune-related markers including CD3, CD4, CD8, Foxp3, 1-selectin arginase, ADAM metallopeptidase domain 17, CD163 and CD45RO within tumor microenvironment were performed in resected gastric tumor specimen. Results: The median value of FoxP3-TSDR and CD3G/CD3D demethylation was 5.8% and 32.3%, respectively. When pTreg/total T cell ratio was divided into eight equal groups from the lowest to the highest value, the extreme pTreg/total T cell ratio of the upper eighth and the lower eighth was significantly associated with lower CD45RO expressed cell counts within tumor microenvironment. In terms of arginase and CD163, inverse results were observed. Patients with extreme pTreg/total T cell ratio had significantly shorter disease-free survival (DFS) and overall survival (OS) compared to the patients with non-extreme pTreg/total T cell ratio. Multivariate analysis which included age, stage, lymphatic invasion, vascular invasion, and perineural invasion as covariates demonstrated that the extreme pTreg/total T cell ratio was an independent predictor for shorter DFS (HR 1.740, 95% CI 1.128 – 2.682; p = 0.012) and OS (HR 1.900, 95% CI 1.175 – 3.070; p = 0.003). Conclusions: Our results suggest that the pTreg/total T cell ratio determined by epigenetic methylation analysis is correlated with specific immune cell infiltration within tumor microenvironment in resected gastric tumors, and gives prognostic information in gastric cancer patients. pTreg/total T cell ratio could be an easy-obtained potential biomarker for prognosis and future immunotherapeutic treatment strategies.