The exocrine pancreas secretes fluid and digestive enzymes in response to parasympathetic release of acetylcholine (ACh) via the vagus nerve and the gut hormone cholecystokinin (CCK). Both secretion of fluid and exocytosis of secretory granules containing enzymes and zymogens are dependent on an increase in the cytosolic [Ca²⁺] in acinar cells. It is thought that the specific spatiotemporal characteristics of the Ca²⁺ signals are fundamental for appropriate secretion and that these properties are disrupted in disease states in the pancreas. While extensive research has been performed to characterize Ca²⁺ signalling in acinar cells, this has exclusively been achieved in ex vivo preparations of exocrine cells, where it is difficult to mimic physiological conditions. Here we have developed a method to optically observe pancreatic acinar Ca²⁺ signals in vivo using a genetically expressed Ca²⁺ indicator and imaged with multi-photon microscopy in live animals. In vivo, acinar cells exhibited baseline activity in fasted animals, which was dependent on CCK1 receptors (CCK1Rs). Both stimulation of intrinsic nervous input and administration of systemic CCK induced oscillatory activity in a proportion of the cells, but the maximum frequencies were vastly different. Upon feeding, oscillatory activity was also observed, which was dependent on CCK1Rs. No evidence of a vago-vagal reflex mediating the effects of CCK was observed. Our in vivo method revealed the spatial and temporal profile of physiologically evoked Ca²⁺ signals, which will provide new insights into future studies of the mechanisms underlying exocrine physiology and that are disrupted in pathological conditions.