It is clear that pancreatic β‐cell dysfunction, including basal hyperinsulinemia and reduced insulin release in response to glucose, is a key determinant of disease progression in type 2 diabetes, but the underlying molecular defects are not known. In diabetes, the expression and function of ryanodine receptor (RyR) Ca²⁺ release channels are reduced. The present studies were undertaken to define the subcellular location and role of RyR in the control of stimulated and basal insulin release from human pancreatic β cells. Using confocal microscopy, we observed RyR immunoreactivity in a vesicular pattern. RyRs did not colocalize with insulin secretory granules but partially colocalized with endosomes. Direct activation with nanomolar concentrations of ryanodine evoked increases in cytosolic Ca²⁺ that were coupled to transient insulin release. Insulin release stimulated by 1 nM ryanodine was sensitive to BAPTA‐AM preincubation but independent of thapsigargin‐sensitive endoplasmic reticulum (ER) Ca²⁺ pools. Blocking RyRs with micromolar concentrations of ryanodine led to BAPTA‐resistant insulin release that was not associated with an increase in cytosolic Ca²⁺, which implicated alterations in luminal Ca²⁺. However, neither Ca²⁺ signals nor insulin release stimulated by glucose was blocked by 10–50 µM ryanodine, which suggests that the CD38/cyclic ADP‐ribose/RyR pathway is not a primary mechanism of glucose action in nontransformed β cells. We provide the first evidence that RyRs directly control insulin secretion in primary β cells. Unexpectedly, stimulation of insulin secretion by ryanodine occurs independently of glucose and by two mechanisms, including a novel cytosolic Ca²⁺‐independent mechanism likely involving changes in Ca²⁺ within the lumens of non‐ER organelles, such as endosomes.