Objective: NAD⁺ is a co-factor and substrate for enzymes maintaining energy homeostasis. Nicotinamide phosphoribosyltransferase (NAMPT) controls NAD⁺ synthesis, and in skeletal muscle, NAD⁺ is essential for muscle integrity. However, the underlying molecular mechanisms by which NAD⁺ synthesis affects muscle health remain poorly understood. Thus, the objective of the current study was to delineate the role of NAMPT-mediated NAD⁺ biosynthesis in skeletal muscle development and function. Methods: To determine the role of Nampt in muscle development and function, we generated skeletal muscle-specific Nampt KO (SMNKO) mice. We performed a comprehensive phenotypic characterization of the SMNKO mice, including metabolic measurements, histological examinations, and RNA sequencing analyses of skeletal muscle from SMNKO mice and WT littermates. Results: SMNKO mice were smaller, with phenotypic changes in skeletal muscle, including reduced fiber area and increased number of centralized nuclei. The majority of SMNKO mice died prematurely. Transcriptomic analysis identified that the gene encoding the mitochondrial permeability transition pore (mPTP) regulator Cyclophilin D (Ppif) was upregulated in skeletal muscle of SMNKO mice from 2 weeks of age, with associated increased sensitivity of mitochondria to the Ca²⁺-stimulated mPTP opening. Treatment of SMNKO mice with the Cyclophilin D inhibitor, Cyclosporine A, increased membrane integrity, decreased the number of centralized nuclei, and increased survival. Conclusions: Our study demonstrates that NAMPT is crucial for maintaining cellular Ca²⁺ homeostasis and skeletal muscle development, which is vital for juvenile survival.