Abstract Objective Osteoarthritis is a condition characterized by articular cartilage degradation. The increased expression of β1,4-Galactosyltransferase-I (β1,4-GalT-I) in the articular cartilage of osteoarthritis patients was related to an inflammatory response. The aim of this study was to elucidate the role of β1,4-GalT-I in osteoarthritis. This study aimed to determine the function of 1,4-GalT-I in osteoarthritis. Methods The osteoarthritis mouse model with the destabilization of the medial meniscus was established by microsurgical technique. Pathological changes in articular cartilage were observed by hematoxylin and eosin staining and safranin O-fast green staining. Quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assays were used to observe mRNA and protein expression, respectively. RNA interactions were verified by a luciferase reporter assay. SA-β-Gal staining was used to assess chondrocyte senescence. Immunofluorescence staining was conducted to observe the localization of Nuclear Factor-kappaB (NF-κB). Results β1,4-GalT-I and microRNA-15a (miR-15a) show high and low expression in the articular cartilage of osteoarthritis, respectively. MiR-15a inhibits the mRNA translation of β1,4-GalT-I. β1,4-GalT-I promotes extracellular matrix degradation, senescence, and NF-κB activation in IL-1β-stimulated chondrocytes, which can be reversed by overexpression of miR-15a. Intra-articular injection of microRNA-15a ameliorates cartilage degeneration by inhibiting β1,4-GalT-I and phosphorylation of NF-κB in vivo. Conclusion The authors clarified that the miR-15a/β1,4-GalT-I axis inhibits the phosphorylation of NF-κB thereby inhibiting extracellular matrix degradation and senescence in chondrocytes to alleviate cartilage degeneration in osteoarthritis. MiR-15a and β1,4-GalT-I may serve as potentially effective targets for the future treatment of osteoarthritis. β1,4GalactosyltransferaseI β14GalactosyltransferaseI βGalactosyltransferaseI β1,4 Galactosyltransferase I β1 4 β β1,4GalTI β14GalTI βGalTI GalT (β1,4-GalT-I response 1,4GalTI 14GalTI GalTI 1,4 1 technique Ofast O fast realtime real time reaction blot enzymelinked enzyme linked respectively assay SAβGal SA Gal FactorkappaB Factor kappaB NFκB. NFκB NF κB . (NF-κB) microRNA15a microRNAa microRNA 15a miR15a miRa miR (miR-15a MiR15a MiRa MiR β1,4GalTI. I. IL1βstimulated ILβstimulated IL 1β stimulated miR15a. 15a. miR-15a Intraarticular Intra vivo miR15a/β1,4GalTI miR15aβ14GalTI miRaβGalTI 15a/β1,4 4GalactosyltransferaseI GalactosyltransferaseI β14 β1, 4GalTI 14 1, (NF-κB βstimulated aβ 15aβ14 15a/β1, 15aβ1 15a/β1 15aβ 15a/β