Nitric oxide (NO) affects fatty acid synthesis and biogenesis of fatty acid consuming mitochondria. However, whether NO generated by the endothelial NO synthase isoform (eNOS) has significant impact on the synthesis and deposition of fat in liver remained unclear. We analyzed the quantity and distribution of mitochondria and fat in liver of wild-type (WT) mice and mice lacking eNOS (eNOS-KO). The livers of eNOS-KO mice contained tenfold more fat close (zone 1) and twenty fold more distal (zone 3) to the artery. The fat was deposited as droplets co-localized with mitochondria. Additionally, the livers of eNOS-KO mice contained 1.5-fold more homogenously distributed glycogen. No difference in the quantity of mitochondria was found between liver homogenates of eNOS-KO mice and WT animals. Mitochondria from liver homogenates of eNOS-KO mice exhibited a higher ratio of citrate synthase (CS) and NADH-cytochrome c oxidoreductase (KI+III) activity. We conclude that lack of eNOS-derived NO stimulates citrate- and lipid synthesis in liver thus contributing to the development of overweight. In support of this view, more visceral fat and 70% higher body weight was determined in one year old eNOS-KO mice in comparison to WT animals.