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Sunburn is one of the most common skin lesions caused by excessive UV exposure, and its incidence is highly correlated with the risks of skin cancer. A variety of drugs including corticosteroids and NSAIDs have been developed to treat acute sunburn, however, they have raised severe concerns such as poor healing efficacy and long recovery time. We have for the first time extracted non-denatured type I collagen from yak hide, which displays a canonical triple helical structure with melting temperature of 42.7 °C. The highly pure yak collagen type I (YCI) self-assembles to form well-ordered nanofibers with periodic d-bands. YCI is highly biocompatible, and it significantly promotes the proliferation and adhesion of HFF-1 cells. The sunburn healing effects of YCI has been investigated using acute skin injury mouse model. Histological analysis shows that 4 days’ treatment of YCI has resulted in the recovery of sunburned mice skin to a healthy state, indicated by pronounced acceleration of epithelization and collagen deposition. The collagen volume fraction as well as the hydroxyproline (Hyp) content of YCI-treated sunburned skin have been found to be greatly increased, confirming the enhanced regeneration of collagen. YCI creams and dressings have also shown superior healing capacity of sunburn by remarkably shortening the recovery time. Notably, the denatured collagen-targeted staining results indicated a large quantity of denatured collagen in sunburned mice, which became substantially reduced after the YCI treatment. FITC-labeled YCI has been further found to penetrate into the dermis of sunburned mice. The highly biocompatible and bioactive non-denatured YCI provides an improved treatment of sunburn, indicating very promising applications of YCI in cosmetics and dermatology.