Headache, muscle aches and chest pain of mild to medium intensity are among the most common clinical symptoms in moderate Staphylococcus aureus infections, with severe infections usually associated with worsening pain symptoms. These nociceptive responses of the body raise the question of how bacterial infection impinges on the nervous system. Does S. aureus, or its released virulence factors, act directly on neurones? To address this issue, we evaluated the potential effects on neurones of certain bi‐component leukotoxins, which are virulent factors released by the bacterium. The activity of four different leukotoxins was verified by measuring the release of glutamate from rat cerebellar granular neurones. The bi‐component γ‐haemolysin HlgC/HlgB was the most potent leukotoxin, initiating transient rises in intracellular Ca²⁺ concentration in cerebellar neurones and in primary sensory neurones from dorsal root ganglia, as probed with the Fura‐2 Ca²⁺ indicator dye. Using pharmacological antagonists of receptors and Ca²⁺ channels, the variations in intracellular Ca²⁺ concentration were found independent of the activation of voltage‐operatedCa²⁺ channels or glutamate receptors. Drugs targeting Sarco‐Endoplasmic Reticulum Ca²⁺‐ATPase (SERCA) or H⁺‐ATPase and antagonists of the store‐operated Ca²⁺ entry complex blunted, or significantly reduced, the leukotoxin‐induced elevation in intracellular Ca²⁺. Moreover, activation of the ADP‐ribosyl cyclase CD38 was also required to initiate the release of Ca²⁺ from acidic stores. These findings suggest that, prior to forming a pore at the plasma membrane, leukotoxin HlgC/HlgB triggers a multistep process which initiates the release of Ca²⁺ from lysosomes, modifies the steady‐state level of reticular Ca²⁺ stores and finally activates the Store‐Operated Calcium Entry complex.