A nonsense polymorphism in the ACTN3 gene (R577X, rs1815739) results in the loss of the fast skeletal muscle fiber protein α-actinin-3 in an estimated 1.5 billion humans worldwide. Homozygosity for this common polymorphism (ACTN3 577XX) does not cause disease but is detrimental to sprint performance in elite athletes. Recently, we reported that ACTN3 577XX humans and Actn3 knockout mice show improved cold tolerance. This was not due to an increase in brown adipose tissue activity or a greater muscle shivering response, but was associated with an increased abundance of slow myosin heavy chain muscle fibers in XX individuals, which in turn increased muscle tone to improve cold tolerance. Using the Actn3 knockout mouse we have now analyzed the molecular and physiological impacts of acute cold exposure (4oC, for 5 hours) on skeletal muscle. In addition, we used statistical inference of publicly available archaic (Neanderthal and Denisovan) and modern human DNA data to estimate the age of the ACTN3 X-allele and to identify a novel haplotype associated with this variant that provides support for genetic selection in some populations of modern humans. Our results indicate that skeletal muscle of Actn3 knockout mice exhibit higher mitochondrial oxidative phosphorylation abundance and activity than wild-type controls. Following acute cold exposure, we identified reductions in gene ontology pathways associated with metabolism and protein breakdown, which impact muscle mass loss and improved muscle fatigue resistance. Together this provides further evidence for improved cold tolerance in Actn3 knockout mice and ACTN3 577XX individuals. Furthermore, we confirm that the X-allele first appeared in modern humans ~135,000 years ago, with this variant increasing in frequency in European and Asian populations from as early as 42,000 years ago, which aligns with modern human migration out of Africa. Taken together these data provide additional molecular and functional evidence for a protective advantage of α-actinin-3 deficiency following cold exposure, highlighting the importance of skeletal muscle in mammalian thermogenesis and its potential role in the evolution of modern-day humans.