The development of testicular inflammation affects the normal male reproductive function. The proteasome activator complex subunit 3 (REGγ) has been suggested to regulate experimental colitis. However, to the best of our knowledge, a potential association between REGγ and testicular inflammation has not been demonstrated. The present study successfully established inflammatory models in C57 mice, primary Leydig cells and the TM3 cell line. It was observed that the absence of REGγ conveyed a significantly protective effect toward testosterone secretion in Leydig cells. REGγ deficiency significantly decreased the expression levels of phosphorylated transcription factor p65 and inflammatory factors in testis tissues, primary Leydig cells and the TM3 cell line. Inflammation also upregulated the expression levels of REGγ. Furthermore, the degradation of the nuclear factor light‑chain‑enhancer of activated B cells (NF‑κB) inhibitor ε (IkBε) signaling pathway regulated REGγ and NF‑κB expression. Double knockdown of REGγ and IkBε restored the response in wild‑type cells to LPS‑induced inflammation. In summary, these results demonstrated that REGγ regulates NF‑κB activity by specifically degrading IkBε to regulate inflammation in testicular Leydig cells.