BACKGROUND/OBJECTIVE: Converging evidence in recent years suggests growth chondrocytes, involved in the integral process of endochondral bone formation and fracture healing, exhibit a dynamic bioenergetic profile despite residing in the nutrient poor cartilaginous environment. Specifically, chondrocytes show an increased dependence on mitochondrial derived oxidative phosphorylation during differentiating, collagen product, but to a differing extent when mineralizing. Therefore, quantitative analysis of mitochondrial dynamics during these varying processes serves to corroborate existing metabolic studies and further elucidate the role of oxidative metabolism during the endochondral process. METHODS: The murine chondroprogenitor cell line ATDC5 was used, and groups were cultured in differentiating, collagen promoted, and mineralizing conditions. Fluorescence confocal 3D image acquisition and bioimaging analysis was used to quantify changes in mitochondrial volume and branch length per mitochondria along with organization and colocalization changes of the actin cytoskeleton to mitochondria in the various conditions over 21 days. RESULTS: We showed that chondrocyte differentiation resulted in significantly increased mitochondrial volume and fusion when compared to non-differentiating groups, and in collagen promoted groups, mitochondrial volume was significantly higher. Additionally, we showed that the process of mineralization resulted in a significant decrease in mitochondrial volume and branch length per mitochondria by day 21 of the experiment. Finally, colocalization analyses of the actin cytoskeleton to mitochondria showed significantly increased overlap in non-differentiating cells when compared to differentiating conditions. CONCLUSIONS: These findings suggest that collagen production is likely an energetically taxing process and mineralization does not heavily rely on oxidative metabolism. Furthermore, the actin cytoskeleton likely plays a role in mitochondrial remodeling that coincides with mitochondrial fission and fusion; increased fission is associated with actin accumulation to mitochondria and fusion is associated with actin disassociation from the outer mitochondrial membrane.