OBJECTIVES: This study was performed to evaluate the burn wound-healing efficacy of crocodile oil from Crocodylus siamensis by employing deep second-degree burns in a Wistar rat model. METHODS: Twenty-four rats were assigned equally into four groups using a random-number table, and two burns were created on the dorsum of each animal except for the sham group. The three treatment groups received with saline solution (12 burns, served as negative control), silver sulfadiazine (12 burns, served as positive control), or crocodile oil (12 burns). Silver sulfadiazine cream was used as standard care, and the treatments were repeated twice daily for 28 days. After day 28 the animals were euthanized and the wounds were removed for quantitative real-time polymerase chain reaction, histologic, and immunohistochemical study. RESULTS: Crocodile oil accelerated the wound-healing process as indicated by a significant decrease in wound closure time in comparison to the burn control and silver sulfadiazine treatment groups. Histologic results showed well-organized and distributed skin structure and collagen deposition in the animals treated with crocodile oil. Transforming growth factor-β1 (TGF-β1), a key cytokine promoting scarring, was also observed to play a role in the burn wound healing. Immunohistochemical staining results showed the negative expression of TGF-β1 and Smad3 in the 28-days-postburn skin of crocodile oil group versus positive in the epidermis of burn controls. Compared to the burn control group, expressions of TGF-β1 and Smad3 mRNA decreased significantly (p < 0.01) in the 28-days-postburn skin of the crocodile oil group. CONCLUSIONS: Our results showed that crocodile oil could enhance cutaneous burn wound healing and reduce scar formation in rats, which might be related to TGF-β1/Smad3 signaling.