Adipose triglyceride lipase (ATGL) catalyzes the first step of lipolysis of cytoplasmic triacylglycerols in white adipose tissue (WAT) and several other organs. We created adipose-specific ATGL-deficient (ATGLAKO) mice. In these mice, in vivo lipolysis, measured as the increase of plasma nonesterified fatty acid and glycerol levels after injection of a β3-adrenergic agonist, was undetectable. In isolated ATGLAKO adipocytes, β3-adrenergic-stimulated glycerol release was 10-fold less than in controls. Under fed conditions, ATGLAKO mice had normal viability, mild obesity, low plasma nonesterified fatty acid levels, increased insulin sensitivity, and increased daytime food intake. After 5 h of fasting, ATGLAKO WAT showed phosphorylation of the major protein kinase A-mediated targets hormone-sensitive lipase and perilipin A and ATGLAKO liver showed low glycogen and triacylglycerol contents. During a 48-h fast, ATGLAKO mice developed striking and complex differences from controls: progressive reduction of oxygen consumption, high respiratory exchange ratio, consistent with reduced fatty acid availability for energy production, lethargy, hypothermia, and undiminished fat mass, but greater loss of lean mass than controls. Plasma of 48 h-fasted ATGLAKO mice had a unique pattern: low 3-hydroxybutyrate, insulin, adiponectin, and fibroblast growth factor 21 with elevated leptin and corticosterone. ATGLAKO WAT, liver, skeletal muscle, and heart showed increased levels of mRNA related to autophagy and proteolysis. In murine ATGL deficiency, adipose lipolysis is critical for fasting energy homeostasis, and fasting imposes proteolytic stress on many organs, including heart and skeletal muscle.