Mesenchymal stem cells (MSCs) are candidates for the regeneration of articular cartilage as they possess the potential for chondrogenic differentiation. MSCs are easily obtained and expanded in vitro. Specific microRNAs (miRNAs) that regulate chondrogenesis have yet to be identified and the mechanisms involved remain to be defined. The miRNAs regulate biological processes by binding target mRNA to reduce protein synthesis. In this study, we show that expression of miR-99a and miR-125b-3p were increased during early chondrogenic differentiation of MSCs (rMSCs) derived from the Norwegian brown rat (Rattus norvegicus). MiR-99a knockdown promoted proteoglycan deposition and increased the expression of ACAN and COL2A1 during early chondrogenic differentiation. MiR-99a knockdown promoted early chondrogenic differentiation of rMSCs. A dual-luciferase reporter gene assay showed that miR-99a targeted a putative binding site in the 3'-UTR of bone morphogenetic protein (BMP) receptor type 2 (BMPR2). Overexpression of miR-99a reduced the expression levels of BMPR2 protein. The expression of total p38 and p-p38 increased at 7 and 14 days during early chondrogenic differentiation of rMSCs. Reduction in levels of total p38 and p-p38 protein followed miR-99a overexpression during early chondrogenic differentiation of rMSCs. BMPR2 silencing reversed the effects of miR-99a inhibition on proteoglycan deposition and protein expression of ACAN, COL2A1, total p38 and p-p38 during early chondrogenic differentiation of rMSCs. In conclusion, the findings of these in vitro studies in rat MSCs support a role for miR-99a as a negative regulator of early chondrogenic differentiation by directly targeting the BMPR2 gene at an early stage.