ATP has been previously identified as an autocrine signaling factor that is co-released with insulin to modulate and propagate β-cell activity within islets of Langerhans. Here, we show that β-cell activity and insulin secretion essentially rely on the presence of extracellular ATP. For this, we monitored changes of the intracellular Ca concentration ([Ca ] oscillations) as an immediate read-out for insulin secretion in live cell experiments. Extensive washing of cells or depletion of extracellular ATP levels by recombinant apyrase reduced [Ca ] oscillations and insulin secretion in pancreatic cell lines and primary β-cells. Following ATP depletion, [Ca ] oscillations were stimulated by the replenishment of ATP in a concentration-dependent manner. Inhibition of endogenous ecto-ATP nucleotidases increased extracellular ATP levels, along with [Ca ] oscillations and insulin secretion, indicating that there is a constant supply of ATP to the extracellular space. Our combined results demonstrate that extracellular ATP is essential for β-cell activity. The presented work suggests extracellular ATPases as potential drug targets for the modulation of insulin release. We further found that exogenous fatty acids compensated for depleted extracellular ATP levels by the recovery of [Ca ] oscillations, indicating that autocrine factors mutually compensate for the loss of others. Thereby, our results contribute to a more detailed and complete understanding of the general role of autocrine signaling factors as a fundamental regulatory mechanism of β-cell activity and insulin secretion.