Collagen formation is used as a crucial indicator of tenogenic differentiation of human tendon derived stem cell (hTDSC). Early growth response-1(egr1), a transcriptional factor, has been demonstrated to regulate tendon differentiation and promote tendon repair. Considering that the therapeutic options for tendon injuries remain limited, investigating the regulation of egr1 could facilitate the understanding of tendon development at molecular level so as to find a promising therapeutic target. MicroRNAs (miRNA) have been considered as epigenetic regulators to mediate multiple biological activities including stem cell differentiation. In the present study, biological experiments confirmed the prediction that miR124–3p (miR124) could have direct binding with egr1. We also found that miR124 suppressed collagen formation during the tendon differentiation of hTDSC while anti-miR124 promoted it. Furthermore, egr1 knockdown abolished the promotive effect of anti-miR124, suggesting that miR124 prevents tendon differentiation via suppressing egr1 expression. Therefore, miR124 may be a promising therapeutic target for tendon injury.