We evaluated the role of the CXCL12/CXCR4 (C-X-C motif chemokine ligand 12/C-X-C chemokine receptor type 4) axis in aggrecanase-mediated cartilage degradation, and explored the underlying mechanism in a post-traumatic osteoarthritis rat model. Expression of CXCL12/CXCR4 and ADAMTS-5 was analyzed in the knees of osteoarthritic and non-arthritic rats using Western blot, ELISA, immunohistochemistry and immunofluorescence. Rodent studies were performed using Sprague-Dawley rats, with animals divided into three groups: Destabilization of the medial meniscus/AMD3100-treated (DMM/AMD3100-treated), DMM/PBS-treated, and sham controls. Rats were sacrificed after eight weeks, and samples were collected for histology and immunohistochemistry analyses. IL-1-pretreated primary chondrocytes were cultured with untreated control, CXCL12a, siNC + CXCL12a, or siRNA CXCR4 + CXCL12a, and analyzed for expression of relevant markers and cellular pathways. Higher levels of CXCL12 were detected in the knee fluid of osteoarthritic subjects, with strong staining for CXCR4 in chondrocytes and CXCL12 in synoviocytes together with enhanced expression of ADAMTS-5. DMM/AMD3100-treated rats showed a significantly reduced immunological response, with minimal evidence of pathology in both histological and immunohistochemical analyses. Treatment with CXCL12a increased the expression of ACAN, RUNX-2, and ADAMTS-4/5 in IL-1-pretreated primary chondrocytes, together with a decrease in the expression of SOX-9. Molecular analyses revealed strong induction of NF-kappa B activation, along with phosphorylation of MAPKs, and activation of canonical Wnt/beta-catenin signaling. In conclusion, inhibition of SDF-1 alpha/CXCR4 signaling axis was able to inhibit aggrecanase expression and lessen cartilage degeneration in post-traumatic osteoarthritis rats.