Brown adipose tissue (BAT) and beige adipocytes within white adipose tissue (WAT) mediate nonshivering thermogenesis through uncoupling protein 1 (UCP1). Here, we characterized the peptidome of interscapular BAT and inguinal WAT under thermogenic stimulation combining cold exposure and pharmacological activation of PPARγ by pioglitazone. C57BL/6 mice were housed at 21 °C or 7 °C for 15 days and treated with pioglitazone (30 mg/kg/day) or vehicle. Mass spectrometry identified 39 peptides in BAT and 95 in WAT, most derived from hemoglobin alpha and beta subunits and upregulated under thermogenic conditions. Twenty-four peptides were common to both tissues, and eleven upregulated peptides (P1–P11) were further investigated in cultured adipocytes and isolated mitochondria. Peptide P8 increased the expression of thermogenesis and adipogenesis-related genes (UCP1, PGC1α, Cpt1a, and FABP4) in brown adipocytes, while P1 and P11 enhanced these genes in white adipocytes. Peptides P2, P4, P6, and P9 increased mitochondrial oxygen consumption in BAT. Collectively, these findings highlight a novel role of intracellular peptides as modulators of thermogenic activity in brown and beige adipocytes and provide novel insights into their biological and therapeutic relevance.