Calcineurin/NFAT1 signaling pathway plays critical roles in maintaining the homeostasis of articular chondrocytes and in regulating the pathogenesis of osteoarthritis (OA). A few studies demonstrate therapeutic values of ropivacaine (Rop) in OA, but the underlying mechanisms have not been defined. Here, we determined whether Calcineurin/NFAT1 signaling pathway mediates the benefits of Rop to OA. OA rat models were established by a single intra-articular injection of monosodium iodoacetate. The pathophysiology of OA was evaluated by measuring hyperalgesia behavior and the expression of NFAT1, calcineurin, catabolic enzymes in chondrocytes, and chondrogenic markers in affected articular cartilage and primary chondrocyte cultures treated with IL-1β. ROP was applied both in vivo and in vitro to examine its effects on the pathophysiology of OA. Hyperalgesia in OA rats was improved by intra-articular injection of Rop. Moreover, Rop suppressed the overexpression of NFAT1, calcineurin, TNF-α, IL-6, MMP1 and MMP3, and reversed the diminution of collagen II and aggrecan, in affected cartilage of OA rats. Similar effects of Rop were also observed in mouse chondrocyte cultures treated with IL-1β. In in vitro preparations, either activation (by increasing extracellular Ca2+) or inhibition (by cyclosporin A) of calcineurin blocked the effects of Rop. These results suggest that Rop may have therapeutic potential for OA in three aspects: analgesia, anti-inflammation, and anti-degradation of articular cartilage, probably via down-regulating calcineurin/NFAT1 signaling pathway.