A genome-wide association study (GWAS) reported that common variation in the human Niemann-Pick C1 gene ( NPC1) is associated with morbid adult obesity. This study was confirmed using our BALB/cJ Npc1 mouse model, whereby heterozygous mice ( Npc1+/−) with decreased gene dosage were susceptible to weight gain when fed a high-fat diet (HFD) compared with homozygous normal mice ( Npc1+/+) fed the same diet. The objective for our current study was to validate this Npc1 gene-diet interaction using statistical modeling with fitted growth trajectories, conduct body weight analyses for different measures, and define the physiological basis responsible for weight gain. Metabolic phenotype analysis indicated no significant difference between Npc1+/+ and Npc1+/− mice fed a HFD for food and water intake, oxygen consumption, carbon dioxide production, locomotor activity, adaptive thermogenesis, and intestinal lipid absorption. However, the livers from Npc1+/− mice had significantly increased amounts of mature sterol regulatory element-binding protein-1 (SREBP-1) and increased expression of SREBP-1 target genes that regulate glycolysis and lipogenesis with an accumulation of triacylglycerol and cholesterol. Moreover, white adipose tissue from Npc1+/− mice had significantly decreased amounts of phosphorylated hormone-sensitive lipase with decreased triacylglycerol lipolysis. Consistent with these results, cellular energy metabolism studies indicated that Npc1+/− fibroblasts had significantly increased glycolysis and lipogenesis, in addition to significantly decreased substrate (glucose and endogenous fatty acid) oxidative metabolism with an accumulation of triacylglycerol and cholesterol. In conclusion, these studies demonstrate that the Npc1 gene interacts with a HFD to promote weight gain through differential regulation of central energy metabolism pathways.