PRL is a 23 kDa hormone produced primarily in the pituitary that is best recognized for its lactogenic activity. However, PRL is also involved in the regulation of metabolic homeostasis in many species. Studies in rats generally indicate that PRL promotes weight gain, while in humans, the association between PRL and adiposity is unclear. Several mouse models and adipocyte cell lines have been used to study the effects of PRL on growth, adipokine secretion, and lipid and glucose metabolism, but a consensus has not emerged, given limited and often conflicting data. Thus, clarifying the metabolic functions of PRL in the mouse would address many published controversies and contribute to understanding the systems and factors that regulate energy balance. Our hypothesis was that PRL is required for normal weight gain and adiposity in male and non-lactating female mice. We postulated that this may occur by PRL-mediated alterations in adipokine secretion, glucose metabolism, and/or adipose tissue growth and metabolism. To assess the metabolic role of PRL in vivo, we first compared 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 high-fat, low-fat, and standard chow. We found that PRL-deficiency had no effect on the rate of weight gain, body composition, serum lipids, or adiponectin levels in either sex on any diet. Glucose tolerance was slightly impaired in 4-week old male pups, but not in adults or in females at any age. Leptin was elevated in male, but not female PRL-deficient mice on a low-fat diet. We then measured the expression of selected genes in the adipose tissue and liver of PRL-knockout and wild-type mice under fed and fasted conditions. Nearly all genes, including lipoprotein lipase, hormone-sensitive lipase, leptin, adiponectin, and resistin, were unchanged in PRL-deficient mice when compared to wild-type mice. One novel factor, fasting-induced adipocyte factor (FIAF) was upregulated by fasting in both adipose tissue and liver of wild-type mice, but not in male PRL-deficient mice. When serum from these animals was analyzed, two isoforms of FIAF were significantly higher in PRL-deficient male mice than wild-types. To determine the effects of PRL on adipocyte metabolism, we utilized the murine 3T3-L1 cell line, as well as cultured adipose tissue explants. PRL at physiological doses had no effect on preadipocyte proliferation, lipid accumulation, or morphological alterations during differentiation in 3T3-L1 cells. Chronic PRL treatment had inconsistent effects on basal and insulin-stimulated glucose uptake, as well as on glucose transporter 4 (GLUT4) expression in mature 3T3-L1 adipocytes, while short-term PRL treatment had no effect on these parameters. Finally, PRL did not affect basal or stimulated lipolysis in adipose tissue explants from mice, but significantly inhibited glycerol release from both rat and human adipose tissue explants in a dose-dependent manner. In conclusion, PRL likely plays a role in regulating adipokine release and glucose metabolism, but does not affect gross metabolic parameters in mice. Adiposity is a reflection of adipocyte growth and lipid storage, both of which are unaffected by PRL treatment in vitro. The lipolytic actions of PRL vary among species, with no effects observed in mice. Thus, the metabolic functions of PRL in the mouse are limited compared to other species.