Background and Objective: Articular cartilage abnormalities are important factors of skeletal disorders and usually are related to glycosaminoglycans synthesis in the cartilage tissue. Presence of some vitamins, amino acids, and minerals increases cartilage regeneration. In this study, Medicago sativa (Alfalfa), which is high in some vitamins, amino acids and minerals, was used for cartilage healing of the pinna in rabbits, and glycoseaminoglycans (GAGs) of the extracellular matrix (ECM) were investigated histochemically. Materials and Methods: Four holes (4-mm in diameter each) were punched in the medial portion of each pinna of 10 New Zealand male rabbits (randomly divided into 2 groups), after shaving the hair and anesthetization by 10% lidocaine (total of 80 holes). The ears of the experimental group were treated by alcoholic extract of M. sativa, while the control group was treated by normal saline daily. Samples of regenerating tissue from day 0 through day 50 after the punch were prepared for microscopic studies. A color intensity of Alcian blue in pH 1 for sulfated molecules and in pH 2.5 for carboxylated GAGs and toluidine blue (that has a metacromatic reaction with sulfated GAGs) were considered. Results: In all 3 histochemical staining methods, an early high-color intensity of cartilage was observed in the treated group compared with the control. Furthermore, the closure of the cartilage hole in the treated group happened faster. Conclusion: It seems that the M. sativa extract, probably because of its vitamin and amino acid composition, increases chondroblasts activity for secretion of carboxylated and sulfated GAGs in ECM, which leads to a faster cartilage wound healing. Therefore, the plant extract provides a good candidate for future studies on its potential role in wound healing and pharmacological applications.