Severe and prolonged lymphopenia frequently occurs in glioblastoma patients after standard chemoradiotherapy and has been associated with significantly worse survival, but its biological mechanism is not well understood. To address this we performed a correlative study designed to prospectively collect and evaluate peripheral blood of glioblastoma patients treated with chemoradiotherapy using genomic and immune monitoring technologies. The RNA sequencing analysis of the peripheral blood mononuclear cells (PBMC) showed an elevated level of myeloidderived suppressor cells (MDSC) regulatory genes in the lymphopenic patients (LP) when compared to non-lymphopenic patients (NLP) after chemoradiotherapy. Additional analysis using flow cytometry and single-cell RNA sequencing further confirmed increased number of circulating MDSC in LP when compared to NLP after chemoradiotherapy. Preclinical murine models were also established to recapitulate this phenomenon and demonstrated a causal relationship between radiation-induced MDSC and systemic lymphopenia using transfusion and depletion experiments. Pharmacological inhibition of MDSC using arginase-1 inhibitor (CB1158) or phosphodiesterase5 inhibitor (tadalafil) during RT successfully abrogated radiation-induced lymphopenia and improved survival in the preclinical models.