Full-thickness skin wounds, associated with deep burns or chronic wounds pose a major clinical problem. Herein, the development of in situ forming hydrogel using a natural silk fibroin (SF) biomaterial for treating burn wounds is reported. Blends of SF solutions isolated from Bombyx mori and Antheraea assama show inherent self-assembly between silk proteins and lead to irreversible gelation at body temperature. Investigation of the gelation mechanism reveals crosslinking due to formation of β-sheet structures as examined by X-ray diffraction and Fourier transform infrared spectroscopy. The SF hydrogel supports proliferation of primary human dermal fibroblasts and migration of keratinocytes comparable to collagen gel (Col) as examined under in vitro conditions. The SF hydrogel also provides an instructive and supportive matrix to the full-thickness third-degree burn wounds in vivo. A 3-week comparative study with Col indicates that SF hydrogel not only promotes wound healing but also shows transitions from inflammation to proliferation stage as observed through the expression of TNF-α and CD163 genes. Further, deposition and remodeling of collagen type I and III fibers suggests an enhanced overall tissue regeneration. Comparable results with Col demonstrate the SF hydrogel as an effective and inexpensive formulation toward a potential therapeutic approach for burn wound treatment.