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Objective Lubricin, a glycoprotein encoded by the proteoglycan 4 (PRG4) gene, is an essential boundary lubricant that reduces friction between articular cartilage surfaces. The loss of lubricin subsequent to joint injury plays a role in the pathogenesis of post-traumatic osteoarthritis (PTOA). Here we describe the development and evaluation of an adeno-associated virus (AAV)-based PRG4 gene therapy intended to restore lubricin in injured joints. The green fluorescent protein (GFP) gene was inserted the PRG4 gene to facilitate tracing the distribution of the transgene product (AAV-PRG4-GFP) in vivo. Methods Transduction efficiency of AAV-PRG4-GFP was evaluated in joint cells, and the conditioned medium containing secreted PRG4-GFP was used for shear loading/friction and viability tests. In vivo transduction of joint tissues following intra-articular injection of AAV-PRG4-GFP was confirmed in the mouse stifle joint in a surgical model of destabilization of the medial meniscus (DMM), and chondroprotective activity was tested in a rabbit anterior cruciate ligament transection (ACLT) model. Results In vitro studies showed that PRG4-GFP has lubricin-like cartilage binding and anti-friction properties. Significant cytoprotective effects were seen when cartilage was soaked in PRG4-GFP prior to cyclic shear loading (n = 3). Polymerase chain reaction and confocal microscopy confirmed the presence of PRG4-GFP DNA and protein, respectively, in a mouse DMM (n = 3 per group). In the rabbit ACLT model, AAV-PRG4-GFP gene therapy enhanced lubricin expression (p = 0.001 versus AAV-GFP: n = 7-14) and protected the cartilage from degeneration (p = 0.014 versus AAV-GFP: n = 9-10) when treatments were administered immediately post-operation, but efficacy was lost when treatment was delayed for 2 weeks. Conclusion AAV-PRG4-GFP gene therapy protected cartilage from degeneration in a rabbit ACLT model; however, data from the ACLT model suggest that early intervention is essential for efficacy.