Background and objectives: Hepatocyte ultra-structure is influenced by feeding status, circadian rhythm, and zone location. The goal of the present study was to study the effect of overnight fasting on the hepatocyte ultrastructure and the zonal heterogeneity and to discuss the functional correlation. Methods: A total of 14 male albino rats were divided into two groups: negative control group fed ad libitum and overnight fasting rats for 16 hours. The different subcellular structures of both centrilobular and periportal hepatocytes in both control and fasted groups were compared by transmission electron microscopy. Morphometric analysis of the electron micrographs was also done using imageJ software. Results: The lysosomes surface density, mitochondrial volume and surface densities were significantly higher in periportal hepatocytes however surface density of smooth endoplasmic reticulum (SER) and peroxisomes were significantly higher in centrilobular hepatocytes of the control group. Fasting caused a significant decrease in the surface density of rough endoplasmic reticulum and glycogen volume density but with significant increase in SER surface density with more mitochondrial fusion and stronger mitochondrial ER contacts, isolation membranes, and autophagosomes. The zonal differences were maintained after fasting. The organelles appeared normal with no signs of degeneration. Conclusion: The organelles appeared normal with no signs of degeneration and the zonal differences were maintained after fasting. The change in hepatocyte ultrastructure after fasting may be related to autophagy.