Bone marrow mesenchymal stem cells (MSCs) give rise to osteoblasts and adipocytes, with an inverse relationship between the two. The MSCs from protease-activated receptor-2 knockout (PAR2 KO) mice have a reduced capacity to generate osteoblasts. We have recently demonstrated that PAR2 KO primary osteoblastic cultures contain more adipocytes than wildtype (WT) cultures and that PAR2 KO bone marrow stromal cultures contain fewer osteoblastic colonies and more adipocytic colonies than cultures from WT mice. To determine the underlying mechanisms of these effects, PAR2 was stably knocked down in a murine bipotential bone marrow stromal cell line, Kusa 4b10, using lentiviral shRNA constructs (F2rl1 KD cells). In cells cultured under osteogenic conditions, mineralisation assays and Oil-Red-O positive cell counts indicated that PAR2 knockdown decreased mineralisation (61%, p < 0.0001) and increased adipogenesis (210%, p < 0.0001). Quantitative PCR analysis demonstrated that expression of the osteoblastic gene Runx2 was lower (61%, p < 0.01) and expression of the adipocytic genes Pparg and Lpl was greater (57%, p < 0.05 and 23%, p < 0.01, respectively) in F2rl1 KD than in vector control cells. Genes of interest were identified as putative mediators of PAR2’s promotion of osteogenesis and suppression of adipogenesis in a recent RNA-seq investigation; C1qtnf3, Gpr35, Grem1, Snorc and Tcea3 were found to be more highly expressed, and Cnr1, Enpep, Hmgn5, Il6 and Ramp3 were expressed at lower levels, in control than in F2rl1 KD cells. Interleukin-6 (IL-6) levels were ~5-fold higher in medium harvested from F2rl1 KD cells than from control cells (p < 0.0001), and neutralizing anti-IL-6 reduced the number of adipocytes in F2rl1 KD cultures to that of control cultures, without affecting mineralisation. Thus, PAR2 appears to be a mediator of the reciprocal relationship between osteogenesis and adipogenesis, with suppression of Il6 expression contributing to its effect.