Inositol trisphosphate (IP₃)-dependent intracellular Ca²⁺ signaling mediates essential hepatic functions, particularly metabolic processes such as glycogenolysis and bile secretion. However, previous studies have been conducted with in vitro/ex vivo experiments in the absence of hormone, neurotransmitters and cell-cell/organ-organ interaction. To overcome these limitations, we developed an in vivo hepatocyte Ca²⁺ imaging method using two transgenic mouse lines: one expressing the ultrasensitive, ratiometric Ca²⁺ sensor protein, yellow Cameleon-Nano50 , and another co-expressing INPP5A, an IP₃-degrading enzyme that selectively attenuates IP₃-mediated Ca²⁺ signals specifically in hepatocytes. Our in vivo imaging studies under anesthetized laparotomy revealed that hepatocytes maintain elevated basal Ca²⁺ levels and display spontaneous oscillatory Ca²⁺ transients compared to those expressing INPP5A. Furthermore, these signals are likely to be modulated by the autonomic nervous system, suggesting an interplay between neural inputs and hepatocyte Ca²⁺ signaling. Further investigation using our current experimental method will elucidate the detailed signaling dynamics of hepatocyte Ca²⁺ activity and its functional relevance in vivo.