Enteroendocrine cells (EECs) are specialized sensors of luminal forces and chemicals in the gastrointestinal (GI) epithelium that respond to stimulation with a release of signalling molecules such as serotonin (5‐HT). For mechanosensitive EECs, force activates Piezo2 channels, which generate a very rapidly activating and inactivating (∼10 ms) cationic (Na⁺, K⁺, Ca²⁺) receptor current. Piezo2 receptor currents lead to a large and persistent increase in intracellular calcium (Ca²⁺) that lasts many seconds to sometimes minutes, suggesting signal amplification. However, intracellular calcium dynamics in EEC mechanotransduction remain poorly understood. The aim of this study was to determine the role of Ca²⁺ stores in EEC mechanotransduction. Mechanical stimulation of a human EEC cell model (QGP‐1) resulted in a rapid increase in cytoplasmic Ca²⁺ and a slower decrease in ER stores Ca²⁺, suggesting the involvement of intracellular Ca²⁺ stores. Comparing murine primary colonic EECs with colonocytes showed expression of intercellular Ca²⁺ store receptors, a similar expression of IP₃ receptors, but a >30‐fold enriched expression of Ryr3 in EECs. In mechanically stimulated primary EECs, Ca²⁺ responses decreased dramatically by emptying stores and pharmacologically blocking IP₃ and RyR1/3 receptors. RyR3 genetic knockdown by siRNA led to a significant decrease in mechanosensitive Ca²⁺ responses and 5‐HT release. In tissue, pressure‐induced increase in the Ussing short circuit current was significantly decreased by ryanodine receptor blockade. Our data show that mechanosensitive EECs use intracellular Ca²⁺ stores to amplify mechanically induced Ca²⁺ entry, with RyR3 receptors selectively expressed in EECs and involved in Ca²⁺ signalling, 5‐HT release and epithelial secretion.