MicroRNAs are a class of short noncoding RNAs that are involved in various biological processes, including differentiation. MicroRNA-140 (miR-140) has been identified as a cartilage-specific microRNA with several targets involved in cartilage development and homeostasis. The aim of this study was to investigate the expression of miR-140 during chondrogenic differentiation of equine cord blood-derived mesenchymal stromal cells (eCB-MSCs). We demonstrate both that miR-140 is highly expressed in normal equine articular cartilage and that eCB-MSCs express significantly higher levels of this microRNA after 14 days of chondrogenic differentiation. Furthermore, miR-140 expression closely paralleled that of the cartilage-specific transcription factor Sox9, suggesting that miR-140 may be under the transcriptional regulation of Sox9 in these cells. The expression patterns of miR-140 targets the chemokine (CXC motif) ligand 12 (CXCL12), A disintegrin and metalloproteinase with thrombosponin motifs (ADAMTS)-5 and insulin growth factor binding protein 5 (IGFBP5) were also determined; however, only CXCL12 and ADAMTS-5 were repressed while miR-140 expression was upregulated. Together, these studies suggest that miR-140 is an important regulator of cartilage development and homeostasis in eCB-MSCs that may act, in part, through the regulation of CXCL12 and ADAMTS-5.