Rutin is a flavonoid glycoside found in many plants, known for its various pharmacological properties. However, its therapeutic potential is limited by its low water solubility, which restricts its bioavailability. Poloxamer 407 (P407) is a multifunctional excipient, which has proven effective in delivering poorly water-soluble drugs and promoting the healing process. Based on this information, this study investigates the wound healing potential of rutin-loaded P407 hydrogels, as an innovative pharmaceutical formulation. In vitro scratch assays on human keratinocytes revealed that combining rutin with P407 significantly enhances cell proliferation as compared to the single components. Moreover, the proposed hydrogel improved wound healing more effectively in rats than the free drug or the commercial wound dressing DuoDERM. Histological analyses confirmed that the formulation increased epithelialization and collagen synthesis in injured epidermises. These findings provide a strong rationale for the use of rutin-loaded P407 hydrogels for the treatment of skin injuries.