Background <p>The peri-implantation period (days 6–14) is a metabolic checkpoint for embryonic viability, yet melatonin’s role in this phase remains unclear despite its known benefits for early embryogenesis.</p> Results <p>In our in vitro embryo culture model, melatonin was undetectable in embryos during this window in both human and mice, yet MT1/MT2 receptors were expressed. Human metabolomic profiling characterized the dynamic changes of differential metabolites, including 5-methylcytosine, succinic acid, and PC (18:1/18:1), during the peri-implantation period, indicating that development at this stage is highly dependent on cell membrane construction, energy metabolism, and key signaling molecules. Further investigation into the role of melatonin revealed that its treatment reduces tryptophan and L-kynurenine levels while increasing nicotinic acid and enhancing NAD⁺ synthesis. In mice, intraperitoneal melatonin increased NAD⁺ levels in embryos and decidual tissue and delayed age-related NAD⁺ decline in maternal serum.</p> Conclusions <p>Exogenous melatonin acts through its receptors to modulate tryptophan metabolism and NAD⁺ synthesis, demonstrating its potential as a key hormonal regulator of embryonic development and reproductive capacity.</p>