Several clinical studies demonstrated that glucosamine sulfate (GS) is effective in controlling osteoarthritis (OA), showing a structure-modifying action. However, little is known about the molecular mechanism(s) by which GS exerts such action and about the effects of GS at a tissue level on osteoarthritic cartilage and other joint structures. Here we provide mechanistic evidence suggesting that in vitro GS attenuates NF-κB activation at concentrations in the range of those observed after GS administration to volunteers and patients, thus strengthening previous findings. Furthermore, we describe the effects of GS at a tissue level on the progression of the disease in a relevant model of spontaneous OA, the STR/ort mouse. In this model, the administration of GS at human corresponding doses was associated with a significant decrease of OA scores. Histomorphometry showed that the lesion surface was also significantly decreased, while the number of viable chondrocytes within the matrix was significantly increased. GS improved the course of OA in the STR/Ort mouse, by delaying cartilage breakdown as assessed histologically and histomorphometrically.