When attached on a matrix produced by a rat bladder carcinoma cell line (804G matrix), rat pancreatic β-cells spread in response to glucose and secrete more insulin compared with cells attached on poly-L-lysine. The aim of this study was to determine whether laminin-5 and its corresponding cell receptor β1 integrin are implicated in these phenomena. By using specific blocking antibodies, we demonstrated that laminin-5 is the component present in 804G matrix responsible for the effect of 804G matrix on β-cell function and spreading. When expression of two well-known laminin-5 ligands, β1 and β4 integrin, was assessed by Western blot and RT-PCR, only the β1 integrin was detected in β-cells. Anti–β1 integrin antibody reduced the spreading of β-cells on 804G matrix. Blockade of the interaction between β1 integrins and laminin-5 resulted in a reduction in glucose-stimulated insulin secretion. Blocking anti–β1 integrin antibody also inhibited focal adhesion kinase phosphorylation induced by 804G matrix. In conclusion, anti–β1 integrin and –laminin-5 antibodies interfere with spreading of β-cells, resulting in decreased insulin secretion in response to glucose. Our findings indicate that outside-in signaling via engagement of β1 integrins by laminin-5 is an important component of normal β-cell function.