Collagen V is a key matrix protein involved in fibril nucleation and lateral fibril growth during extracellular matrix assembly. Genetic mouse models have been used to investigate the role of collagen V in tendon, which showed deficient mechanical properties and aberrant fibril structure in the absence of collagen V. However, the lasting effects of collagen V deficiency later into adulthood remain unknown, as well as the role of collagen V in maintaining a mature matrix. This study therefore investigated the long-term effects of collagen V reduction on tendon as well as its role in mature tendon matrix in adulthood. Tendon-targeted conditional Col5a1 knockout, which excises Col5a1 alleles early in development, had long-term impact on tendon structure and function in 300-day old mice. Gene expression was altered with differential expression of primarily matrix and matrix remodeling genes. Regional changes in cellular shape and density were consistent with typical behavior in tendinopathy. Fibril diameters were increased due to dysregulated lateral growth. Deficits in mechanical properties indicate a weaker tendon matrix after knockout, although deformation patterns of collagen fibrils were not affected. In contrast, inducing collagen V knockdown in a mature tendon matrix at 120-days old did not cause substantial changes in any of the above-mentioned properties in 300-day old mice. In conclusion, these findings highlight the important function of collagen V in matrix assembly that has lasting effects into later ages, even though collagen V has little role in homeostatic maintenance of a mature tendon matrix.