In addition to promoting insulin secretion, an increase in cytosolic Ca²⁺ triggered by glucose has been shown to be crucial for spreading of beta-cells attached on extracellular matrix (804G matrix). Calpains are Ca²⁺-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca²⁺-induced insulin secretion and spreading of rat pancreatic beta-cells. The results indicate calpain dependency of beta-cell spreading on 804G matrix. Indeed treatment with three distinct calpain inhibitors (ALLN, Calpeptin, EST) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secretory response after 24 hours. In addition, incubation with Calpeptin did not affect insulin secretory response to Mastoparan that acts on exocytosis downstream of intracellular calcium [Ca²⁺]_i. Finally, Calpeptin was shown to affect the [Ca²⁺]_i response to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary beta pancreatic cells. It is therefore suggested that calpain could be a mediator of Ca²⁺-induced-insulin secretion and beta-cell spreading.