Coxsackievirus and adenovirus receptor (CAR) is a junction molecule that expresses on Sertoli and germ cells. It mediates Sertoli-germ cell adhesion and facilitates migration of preleptotene/leptotene spermatocytes across the blood–testis barrier, suggesting that CAR-based cell adhesion and migration are crucial for spermatogenesis. Interferon-gamma (IFNG) and tumor necrosis factor alpha (TNF) are two major cytokines that are elevated during testicular inflammation and cause reduced fertility. We investigated the mechanism by which IFNG and TNF exert their disruptive effects on testicular cell adhesion. We have demonstrated that combined treatment with IFNG and TNF (IFNG TNF) exerts a synergistic effect by downregulating CAR mRNA and protein levels. Immunofluorescence staining revealed that IFNG TNF treatment effectively removes CAR from the site of cell–cell contact. Using inhibitor and co-immunoprecipitation, we confirmed that IFNG TNF mediates CAR protein degradation via ubiquitin-proteasome and NFKB pathways. Blockage of ubiquitin-proteasome pathway significantly inhibits CAR degradation, as indicated by the reappearance of CAR at the site of cell–cell contact. Additionally, IFNG TNF reduces CAR mRNA via transcriptional regulation. Mutational studies have shown that IFNG TNF-induced CAR repression is achieved by suppression of the basal transcription. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays further confirmed that IFNG TNF treament not only inhibits binding of the basal transcription factors but also promotes binding of NFKB subunits and Sp1 (negative regulators) to the CAR promoter region. Taken together, IFNG TNF treatment significantly downregulates CAR expression, which provides an explanation of how cell sloughing in the epithelium mediates, by loss of CAR-based cell adhesion, during testicular inflammation.