During growth phase, antlers exhibit a very rapid rate of chondrogenesis. The antler is formed from its growth center reserve mesenchyme (RM) cells, which have been found to be the derivatives of paired related homeobox 1 (Prrx1)-positive periosteal cells. However, the underlying mechanism that drives rapid chondrogenesis is not known. Herein, the miRNA expression profiles and chromatin states of three tissue layers (RM, precartilage, and cartilage) at different stages of differentiation within the antler growth center were analyzed by RNA-sequencing and ATAC-sequencing. We found that miR-140-3p was the miRNA that exhibited the greatest degree of upregulation in the rapidly growing antler, increasing from the RM to the cartilage layer. We also showed that Prrx1 was a key upstream regulator of miR-140-3p, which firmly confirmed by Prrx1 CUT&Tag sequencing of RM cells. Through multiple approaches (three-dimensional chondrogenic culture and xenogeneic antler model), we demonstrated that Prrx1 and miR-140-3p functioned as reciprocal negative feedback in the antler growth center, and downregulating PRRX1/upregulating miR-140-3p promoted rapid chondrogenesis of RM cells and xenogeneic antler. Thus, we conclude that the reciprocal negative feedback between Prrx1 and miR-140-3p is essential for balancing mesenchymal proliferation and chondrogenic differentiation in the regenerating antler. We further propose that the mechanism underlying chondrogenesis in the regenerating antler would provide a reference for helping understand the regulation of human cartilage regeneration and repair.