This work aimed to study the dysregulated network of galectins in OA chondrocyte pellets, and to assess whether their recently discovered activity as molecular switches of functional biomarkers results in degradation of extracellular matrix in vitro. Scaffold-free 3D pellet cultures were established of human OA chondrocytes. Expression and secretion of galectin(Gal)-1, -3, and -8 were monitored relative to 2D cultures or clinical tissue sections by RT-qPCR, immunohistochemistry and ELISAs. Exposure of 2D and 3D cultures to an in vivo-like galectin mixture (Gal-1 and Gal-8:​ 5 µg/ml, Gal-3:​ 1 µg/ml) was followed by the assessment of pellet size, immunohistochemical matrix staining, and/or quantification of MMP-1, -3, and -13. Application of inhibitors of NF-κB activation probed into the potential of intervening with galectin-induced matrix degradation. Galectin profiling revealed maintained dysregulation of Gal-1, -3, and -8 in pellet cultures, resembling the OA situation in situ. The presence of the galectin mixture promoted marked reduction of pellet size and loss of collagen type II-rich extracellular matrix, accompanied by the upregulation of MMP-1, -3, and -13. Inhibition of p65-phosphorylation by caffeic acid phenethyl ester effectively alleviated the detrimental effects of galectins, resulting in downregulated MMP secretion, reduced matrix breakdown and augmented pellet size. This study suggests that the dysregulated galectin network in OA cartilage leads to extracellular matrix breakdown, and provides encouraging evidence of the feasible inhibition of galectin-triggered activities. OA chondrocyte pellets have the potential to serve as in vitro disease model for further studies on galectins in OA onset and progression.