Background: Typical vivarium temperatures (20-26°C) induce facultative thermogenesis in mice, a process attributable in part to uncoupling protein-1 (UCP1). The impact of modest changes in housing temperature on whole body and adipose tissue energetics in mice remains unclear. Here, we determined the effects of transitioning mice from 24°C to 30°C on total energy expenditure and adipose tissue protein signatures. Methods: C57BL/6J mice were housed at 24°C for two weeks and then either remained at 24°C (n=16 per group, 8M/8F) or were transitioned to 30°C (n=16 per group, 8M/8F) for 4 weeks. Total energy expenditure and its components were determined by indirect calorimetry. Interscapular brown adipose tissue (iBAT) and inguinal white adipose tissue (iWAT) proteins were quantified by western blot and quantitative proteomics. Results: Transitioning from 24°C to 30°C reduced total energy expenditure in both male (-25%) and female (-16%) mice, which was attributable to 36% and 40% decreases in basal energy expenditure in males and females, respectively. Total iBAT UCP1 protein content was 50% lower at 30°C compared to 24°C, whereas iWAT UCP1 protein content was similar between conditions. iBAT UCP1 protein content remained 20-fold greater than iWAT at 30°C. 183 and 41 proteins were differentially expressed between 24°C and 30°C in iBAT and iWAT, respectively. 257 iWAT proteins differentially expressed between sexes at 30°C were not differentially expressed at 24°C. Summary: 30°C housing lowers total energy expenditure of mice when compared to an ambient temperature (24°C) that falls within the National Research Council's guidelines for housing laboratory mice. Lower iBAT UCP1 content accompanied chronic housing at 30°C. Further, housing temperature influences sexual dimorphism in the iWAT proteome. These data have implications regarding the optimization of preclinical models of human disease.