Despite numerous reports of relationships between weight gain and butyrylcholinesterase (BChE), this enzyme's role in the genesis of obesity remains unclear, but recent research points to strong links with ghrelin, the "hunger hormone." The availability of BChE knockout (KO) mice provides an opportunity to clarify the causal relationship between BChE and obesity onset. We now find that young KO mice have abnormally high plasma ghrelin levels that slowly decline during long-term high-fat feeding and ultimately drop below those in wild-type mice. On such a diet, the KO mice gained notably more weight, more white fat, and more hepatic fat than wild-type animals. In addition to a greater burden of hepatic triglycerides, the livers of these KO mice show distinctly higher levels of inflammatory markers. Finally, their energy expenditure proved to be lower than in wild-type mice despite similar activity levels and increased caloric intake. A gene transfer of mouse BChE with adeno-associated virus vector restored nearly all aspects of the normal phenotype. Our results indicate that BChE strongly affects fat metabolism, has an important impact on fat accumulation, and may be a promising tool for combating obesity.