The brown adipose tissue is a potential target for interventions aimed at treating obesity and other metabolic disorders. Both genetic and environmental factors are known to regulate brown adipose tissue function and exploring the interaction between these factors could unveil new mechanisms involved in the regulation of thermogenesis. In this study, we evaluated three genetically distinct mice strains submitted to two environmental factors known to modulate brown adipose tissue function, namely, cold exposure and the consumption of a high- fat diet. The comparison of Balb/c, C57BL/6, and Swiss mice revealed that Balb/c mice were the most glucose- tolerant and the most cold-tolerant. In addition, Balb/c presented the greatest brown adipose tissue oxygen consumption, which was independent of differences in uncoupling protein 1 expression and function. The search for uncoupling protein 1-independent mechanisms that could explain the greatest cold tolerance of Balb/c mice resulted in the identification of the N-acyl amino acid regulator, PM20D1, which had a greater gene expression in the brown adipose tissue of Balb/c mice as compared to the other two strains. The immunoneutralization of PM20D1 in Balb/c mice, resulted in increased blood glucose levels and worsening of cold tolerance. In addition, the in silico knockout of Pm20d1 impacted several metabolic processes, including thermogenesis, glucose tolerance, and insulin sensitivity. In conclusion, Balb/c mice are protected from glucose and acute cold intolerance, independently of the diet. We propose that PM20D1, in an uncoupling protein 1-independent fashion, can have an important role in this protection.