Nicotinamide (NAM) is a widely utilized compound in cell culture systems, yet its role during the morula-to-blastocyst transition remains underexplored. This study investigates the effects of NAM supplementation during both in vitro maturation (IVM) of oocytes and late-stage embryo culture (IVC3; the morula stage) on blastocyst development, metabolic flux, mitochondrial bioenergetics, and pluripotency of cells. Bovine oocytes were subjected to dual NAM treatment during IVM and IVC3 and its impact was assessed through cleavage and blastocyst development rates, mitochondrial membrane potential (ΔΨm), and the expression of key metabolic and pluripotency markers using RT-qPCR and immunofluorescence. Additionally, molecular docking was performed to evaluate NAM's interaction with Zonula Occludens-1 (ZO-1) protein. Dual NAM administration significantly increased both blastocyst formation and hatching rates. Computational modeling revealed a strong binding affinity (-6.44 kcal/mol) between NAM and the ZO-1 protein, associated with the morula-to-blastocyst transition. Quantitative RT-PCR analysis showed upregulation of genes related to NAD + biosynthesis (NAMPT, MDH1), glycolysis (PFK1), glycogenesis (GSK-3A), and mitochondrial bioenergetics (SDHA, ND2, ATPase8, TFAM) in NAM-treated group. Additionally, mitochondrial profiling demonstrated enhanced polarization, and OCT4 expression was elevated in NAM-treated embryos. These findings underscore NAM's potential role in enhancing morula-to-blastocyst transition, improving embryonic development through metabolic and mitochondrial regulation, as well as pluripotency factor enhancement.