Runx2 is a transcription factor prerequisite for chondrocyte maturation and osteoblast differentiation. We tested the hypothesis that Runx2 is responsible for signaling chondrocyte maturation and endochondral ossification in the condyle during mandibular advancement. Fifty 35-day-old Sprague-Dawley rats were fitted with functional appliances for 3, 7, 14, 21, and 30 days. Experimental animals with 50 matched controls were labeled with bromodeoxyuridine for evaluation of the invasion of chondroclasts and osteoblasts into condylar cartilage. Mandibular advancement elicited Runx2 expression in condylar cartilage, and subsequently led to an expansion of type X collagen domain in the hypertrophic layer. Stronger Runx2 mRNA signals in subchondral bone corresponded with the increase in the recruitment of osteoblasts and chondroclasts, which preceded the increase of new bone formation in the condyle. Thus, Runx2 mediates chondrocyte terminal maturation and endochondral ossification in the mandibular condyle in response to mandibular advancement.