This study aimed to explore the efficacy and optimal delivery time of human umbilical cord mesenchymal stem cells (hUC-MSCs) in treating collagenase-induced Achilles tendinopathy. Achilles tendinopathy in rats at early or advanced stages was induced by injecting collagenase I into bilateral Achilles tendons. A total of 28 injured rats were injected with a hUC-MSC solution or normal saline into bilateral tendons twice and sampled after 4 weeks for histological staining, gene expression analysis, transmission electron microscope assay and biomechanical testing analysis. The results revealed better histological performance and a larger collagen fiber diameter in the MSC group. mRNA expression of TNF-α, IL-1β and MMP-3 was lower after MSC transplantation. Early MSC delivery promoted collagen I and TIMP-3 synthesis, and strengthened tendon toughness. hUC-MSCs demonstrated a therapeutic effect in treating collagenase-induced Achilles tendinopathy, particularly in the early stage of tendinopathy.