Prolactin (PRL), best recognized for its lactogenic activity, is also involved in the regulation of metabolic homeostasis in both mammalian and non-mammalian species. Although several mouse models have been used to study the metabolic functions of PRL, a clear-cut consensus has not emerged given the limited and often conflicting data. To clarify the role of PRL in metabolic homeostasis in males and non-lactating females, we used the PRL-deficient mouse. Our objectives were to compare: (1) weight gain, (2) body composition, (3) serum lipid profile, (4) circulating leptin and adiponectin levels, and (5) glucose tolerance in PRL-knockout, heterozygous, and wild-type mice maintained on standard chow, high-fat, or low-fat diets. In addition, we compared the lipolytic actions of PRL using adipose tissue explants from mice, rats and humans. We are reporting that PRL-deficiency does not affect the rate of weight gain, body composition, serum lipids or adiponectin levels in either sex on any diet. Glucose tolerance was slightly impaired in very young PRL-knockout male pups, but not in adults or in females at any age. Leptin was elevated in male, but not female, PRL-knockout mice maintained on LF diet. PRL did not affect lipolysis in adipose tissue explants from mice, but significantly inhibited glycerol release from both rat and human adipose explants in a dose-dependent manner. We conclude that PRL deficiency has negligible gross metabolic effects in mice.