Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS), primary effusion lymphoma(PEL), multicentric Castleman disease, and other tumors. Progression of KS is dictated by an aberrant production of inflammatory cytokines and increase in KSHV infection of cells. In this study, we analyzed the effect of cigarette smoke concentrate (CSC) on KSHV infection of human foreskin fibroblasts (HFF) using real time quantitative RT-PCR. Our results demonstrated that the CSC-treated cells supported 50% lower infection of KSHV when compared to the untreated cells. Radiolabeled-binding assays indicated that CSC inhibited KSHV infection of cells at a post attachment stage of entry. Taken together, we report for the first time the ability of CSC to specifically inhibit KSHV infection of cells.