Obesity is one of the major health problems of our times. Elucidating the signaling mechanisms by which high‐fat caloric diet induces obesity is critical for the understanding of this condition and for the development of therapeutic strategies for its treatment. Here, we demonstrate a novel role for protein CD38 as a regulator of body weight during a high‐fat diet. CD38 is a ubiquitous enzyme that catalyzes the synthesis of second messengers and has been implicated in the regulation of a wide variety of signaling pathways. We report that CD38‐deficient mice are pro‐tected against high‐fat diet‐induced obesity owing to enhanced energy expenditure. In fact, calorimetric studies indicate that CD38‐deficient animals have a higher metabolic rate compared to control mice. Analysis of the mechanism revealed that this resistance to diet‐induced obesity is mediated at least in part via a NAD‐dependent activation of SIRT‐PGC1α axis, a well‐established cascade, involved in the regulation of mito‐chondrial biogenesis and energy homeostasis. Thus, together these results identify a novel pathway regulating body weight and clearly show that CD38 is a nearly obligatory component of the cellular cascade that led to diet‐induced obesity.—Barbosa, M. T. P., Soares, S. M., Novak, C. M., Sinclair, D., Levine, J. A., Aksoy, P., Chini, E. N. The enzyme CD38 (a NAD glycohydrolase, EC 3.2.2.5) is necessary for the development of diet‐induced obesity. FASEB J. 21, 3629–3639 (2007)