Dietary fat composition is a potential major factor affecting energy metabolism. Crocodile oil (CO) is rich in mono- and poly-unsaturated fatty acids exhibiting anti-inflammatory and healing properties. This study investigated different levels of CO consumption on alterations and expression of proteins involved in energy metabolism in rats. Twenty-one male Sprague-Dawley rats were divided into three groups and administered sterile water (N) or different doses of CO [1% or 3% (v/w) CO] orally once daily for 8 weeks. Body weight gain, food intake, energy intake, blood lipid profiles, and serum energy-related metabolites were determined. The serum proteome was analyzed using shotgun proteomics, and the functions of several candidate proteins were classified using PANTHER software. There were no significant differences in body weight or energy intake were observed between groups. However, both CO-treated groups showed significantly decreased serum triglyceride (TG) levels (p < 0.05). Moreover, post-treatment serum TG levels in the 1%CO group were significantly lower than pre-treatment compared with other groups. The serum oxaloacetate level was also significantly higher in both CO groups than in the N group. The proteomic analysis classified 4,525 serum proteins and revealed more unique proteins involved in cellular metabolic activity in both CO-treated groups than in the N group. Self-organizing tree algorithm clustering of 295 shared differentially expressed proteins in both CO-treatment groups showed that upregulated hyper-expressed protein clusters in both CO groups were associated with catalytic activity and molecular activity on the same levels. CO simultaneously enhances energy metabolism and improves lipid profiles.