Abstract: In this study, we aimed to investigate the absorption pathways and efficacy of Gold butterfly (a type of collagen derived from a specific source, herein referred to as Gold butterfly collagen) peptides in mice. Gold butterfly collagen peptides were extracted and purified using an in vitro digestion method, and their amino acid composition was analyzed. Subsequently, isotope-labeled peptide segments ¹³C₅-¹⁵N₁-Pro were prepared via solid-phase synthesis to replace all homologous amino acids in the target peptide segments, and peptides were administered to C57BL/6 mice via gavage. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed to dynamically monitor the metabolic distribution of labeled proline in various tissues and organs over 24 hours. The characteristic predominant amino acids of Gold butterfly collagen peptides were glycine (20.80%), proline (10.72%), and alanine (10.60%). The small intestine was the primary organ absorbing Gold butterfly peptides, with uptake exceeding 50% across all time points. Skin absorption increased to 11.71% at 12 hours, suggesting potential involvement of Gold butterfly peptides in skin tissue repair. Uptake into the stomach, cecum, and colon exhibited dynamic fluctuations, while uptake by the heart and lungs was relatively low. This study demonstrates that Gold butterfly collagen peptide digestion and absorption processes exhibit prominent temporal and organ-specific characteristics, providing theoretical foundation for their nutritional applications.