The ERK–MAPK signaling pathway plays a pivotal role during mesenchymal stem cell (MSC) differentiation. Studies have demonstrated that ERK–MAPK promotes adipogenesis and osteogenesis through the phosphorylation of differentiation-associated transcription factors and that it is the only active signaling in all three lineages (adipogenic, chondrogenic, and osteogenic) during MSC differentiation. Recent studies pointed to the significant roles of microRNA-21 (miR-21) during several physiological and pathological processes, especially stem cell fate determination. The miR-21 expression pattern is also correlated with ERK–MAPK activity. Here, we found that miR-21 expression is elevated and associated with an increased differentiation potential in MSCs during adipogenesis and osteogenesis. The overexpression of miR-21 elevated the expression level of the differentiation-associated genes PPARγ and Cbfa-1 during MSC differentiation, whereas miR-21 knockdown reduced the expression level of both genes. The ERK–MAPK signaling pathway activity had an increasing tendency to respond to miR-21 upregulation and a decreasing tendency to respond to miR-21 down-regulation during the first 4 days of adipogenesis and osteogenesis. Our data indicate that miR-21 modulated ERK–MAPK signaling activity by repressing SPRY2 expression, a known regulator of the receptor tyrosine kinase (RTK) signaling pathway, to affect the duration and magnitude of ERK–MAPK activity. The ERK–MAPK signaling pathway was regulated by Sprouty2 (SPRY2) expression via a miR-21-mediated mechanism during MSC differentiation. J. Cell. Biochem. 114: 1374–1384, 2013. © 2012 Wiley Periodicals, Inc.