Objective: Macrophages play an important part in the pathogenesis of osteoarthritis (OA). Our objective was to determine the effects of α-defensin-1 on macrophage polarization and consequently OA. Methods: OA synovial tissue and synovial fluid were assessed for the presence of M1 (CD68+CD16+CD206-) and M2 (CD68+CD206+CD16-) macrophages by flow cytometry. M0, M1, and M2 macrophages were co-cultured with OA chondrocytes to determine their influence on chondrogenic phenotype. Polarization of THP-1 activated monocytes from M1 to M2 in response to α-defensin-1 was evaluated by flow cytometry, RT-PCR and RNA sequencing. Effects of intra-articular α-defensin-1 in vivo were evaluated in a rat meniscal/ligamentous injury (MLI) model. Results: The quantity of M1 exceeded M2 polarized macrophages in human OA synovial tissue (mean difference 26.1% [13.6-38.6%], P < 0.001) and fluid (mean difference 10.5% [5.0-16.1%], P = 0.003). M1 to M2 polarization in vitro was most effectively promoted with 10 ng/mL α-defensin-1. Compared with untreated macrophages, the α-defensin-1 polarized macrophages modified co-cultured OA chondrocytes from a pro-catabolic state to a pro-anabolic (regenerative-like) state based on expression of COL2A1, ACN, MMP3, MMP13 and ADAMTS5. Intra-articular α-defensin-1 decreased severity of cartilage damage and synovitis in the MLI rat model. RNAseq analyses suggested insulin and Toll-like receptor signaling pathways in the chondroprotective α-defensin-1 mechanism of action. Conclusion: α-defensin-1 promotes M1 to M2 macrophage polarization in vitro, has beneficial effects on chondrocytes indirectly via M2 macrophage polarization, and attenuates the severity of OA in vivo, suggesting it might be a candidate treatment for OA.Keywords: Chondroprotection; Inflammation; Macrophage; Osteoarthritis; Polarization; α-Defensin-1.