Aquaporins (AQPs) are group of membranous water channel proteins that regulates water and small molecule movements through cell membrane. Among this group, aquaporin 8 which express in high abundance in colon, liver and kidneys. With the generation of aquaporin 8 knockout model, C57BL6 mice hepatic tissues were fractionated to purify mitochondrial fractions in both wild and AQP8 knockout model. Tandem mass spectrometric analysis revealed a significant downregulation of glycogen phosphorylase in AQP8 knockout mice. This finding was supported by western blotting. Immune-gold electron microscopy showed mitochondrial cisternae localization of glycogen phosphorylase in higher abundance than in knockout model. Present findings suggest a novel strong association between aquaporin 8 and mitochondrial glycogen phosphorylase which pinpoints indirect involvement of AQP8 in glycogen metabolism.