Selenoprotein S (SelS), a member of the selenoprotein family, is mainly located on the endoplasmic reticulum (ER) membrane. SelS is involved in a variety of biological processes, including oxidative stress, inflammation, glucose metabolism regulation, and ER-associated protein degradation (ERAD). This study was designed to explore the role of SelS in chondrocytes. It was confirmed that SelS is a Se-sensitive selenoprotein in low-selenium rat and cell models. ER stress was not induced in SelS knockdown ATDC5 cells. However, treatment of ATDC5 cells with tunicamycin (Tm), an ER stress inducer, increased the expression of SelS, and knockdown of SelS aggravated ER stress induced by Tm, suggesting that SelS is a regulatory molecule involved in ER stress in chondrocytes. Both osteoarthritis and Kashin-Beck disease are osteochondral diseases associated with hypertrophic chondrocyte abnormalities. Therefore, ATDC5 cells were induced to hypertrophic chondrocytes. SelS was knocked down and RNA sequencing was performed. Bioinformatics analysis of the differentially expressed genes (DEGs) revealed that SelS knockdown affected a variety of biological processes, including cell adhesion, osteoclast differentiation, and extracellular matrix homeostasis. Collectively, this study verified that SelS is sensitive to selenium levels and is an ER stress-responsive molecule. Knocking down SelS can cause abnormal expression of adhesion molecules and matrix homeostasis disorder in hypertrophic chondrocytes.