Globin digest (GD) is a bioactive oligopeptide derived from porcine hemoglobin proteins. GD has been demonstrated to have beneficial effects on improving postprandial hyperlipidemia, hyperglycemia, and liver injury. In this study, we sought to reveal the underlying molecular mechanisms of GD using zebrafish and mouse obesity models. GD suppressed the accumulation of visceral adipose tissue in juvenile zebrafish, and ameliorated several obesity traits in adult obese zebrafish, including dyslipidemia and visceral adiposity. We performed transcriptome analysis by RNA sequencing of GD-treated adult zebrafish and found that GD upregulated UCP1-related pathways. Further, we performed mouse experiments and found that GD intake (2 mg/g body weight/day) showed a mild hypotriglyceridemic effect and improved adipocyte hypertrophy with the upregulation of Ucp1 expression in white adipose tissue at both the mRNA and protein levels. In conclusion, we identified that GD supplementation exerts anti-obesity effects through UCP1 upregulation, further suggesting improvements in energy expenditure.