MiR-140 is a microRNA specially involved in chondrogenesis and osteoarthritis pathogenesis. However, its transcriptional regulation and target genes in cartilage development are not fully understood. Here we detected that miR-140 was uniquely expressed in chondrocyte and suppressed by Wnt/β-catenin signalling. The miR-140 primary transcript was an intron-retained RNA co-expressed with Wwp2-C isoform, which was directly induced by Sox9 through binding to the intron 10 of Wwp2 gene. Knockdown of miR-140 in limb bud micromass cultures resulted in arrest of chondrogenic proliferation. Sp1, the activator of the cell cycle regulator p15^INK4b, was identified as a target of miR-140 in maintaining the chondrocyte proliferation. Collectively, our findings expand our understanding of the transcriptional regulation and the chondrogenic role of miR-140 in chondrogenesis.