• Type V collagen regulates the collagen fibril nanostructure and micromechanics of both the fibrocartilage and hyaline cartilage layers in temporomandibular joint condyle. • Reduction of type V collagen leads to decreased cell density and aberrant cell clustering in both fibrous and hyaline layers. • Loss of type V collagen leads to reduced cell proliferation and β -catenin expression in the fibrous layer, indicating its role in maintaining the progenitor cell niche in condylar cartilage. • Ablation of type V collagen at the post-weaning age results in pronounced thinning of the hyaline layer, highlighting the interplay between type V collagen and mechanoregulation of condylar cartilage growth. • The role of type V collagen in regulating cell fate is specific to the progenitor cells in condylar cartilage, and is absent in knee cartilage. This study queried the role of type V collagen in the post-natal growth of temporomandibular joint (TMJ) condylar cartilage, a hybrid tissue with a fibrocartilage layer covering a secondary hyaline cartilage layer. Integrating outcomes from histology, immunofluorescence imaging, electron microscopy and atomic force microscopy-based nanomechanical tests, we elucidated the impact of type V collagen reduction on TMJ condylar cartilage growth in the type V collagen haploinsufficiency and inducible knockout mice. Reduction of type V collagen led to significantly thickened collagen fibrils, decreased tissue modulus, reduced cell density and aberrant cell clustering in both the fibrous and hyaline layers. Post-natal growth of condylar cartilage involves the chondrogenesis of progenitor cells residing in the fibrous layer, which gives rise to the secondary hyaline layer. Loss of type V collagen resulted in reduced proliferation of these cells, suggesting a possible role of type V collagen in mediating the progenitor cell niche. When the knockout of type V collagen was induced in post-weaning mice after the start of physiologic TMJ loading, the hyaline layer exhibited pronounced thinning, supporting an interplay between type V collagen and occlusal loading in condylar cartilage growth. The phenotype in hyaline layer can thus be attributed to the impact of type V collagen on the mechanically regulated progenitor cell activities. In contrast, knee cartilage does not contain the progenitor cell population at post-natal stages, and develops normal structure and biomechanical properties with the loss of type V collagen. Therefore, in the TMJ, in addition to its established role in regulating the assembly of collagen I fibrils, type V collagen also impacts the mechanoregulation of progenitor cell activities in the fibrous layer. We expect such knowledge to establish a foundation for understanding condylar cartilage matrix development and regeneration, and to yield new insights into the TMJ symptoms in patients with classic Ehlers-Danlos syndrome, a genetic disease due to autosomal mutation of type V collagen. [ABSTRACT FROM AUTHOR]