<p>Clinically relevant concentrations of arsenic trioxide (ATO) induce apoptosis in NB4 cells through a complex, yet poorly defined interplay between endoplasmic reticulum—derived Ca²⁺ signalling and mitochondrial oxidative stress. This study enhances our understanding of these mechanisms by demonstrating that exposure to 1 µM ATO initiates a biphasic Ca²⁺ release: an initial flux from inositol 1,4,5-trisphosphate receptors (IP₃Rs), followed by a secondary release via ryanodine receptors (RyRs). Unlike IP₃R-derived Ca²⁺, the fraction of the cation released through RyRs is subsequently taken up by mitochondria. Notably, IP₃R-derived Ca²⁺ uniquely activates NADPH oxidase 2 (NOX 2), a key event leading to the downstream generation of mitochondrial superoxide&#xa0;(mitoO₂^.−). Importantly, mitochondrial Ca²⁺ accumulation itself is not required for mitoO₂^.− emission. ATO-induced genomic DNA strand breaks are mediated by NOX 2-derived reactive oxygen species (ROS), both directly and indirectly, through the subsequent induction of mitochondrial ROS formation. Furthermore, mitochondrial uptake of RyR-derived Ca²⁺ is essential for triggering the mitochondrial permeability transition and the ensuing apoptotic cell death. Although sodium arsenite elicited comparable effects on Ca²⁺ homeostasis, it promoted mitoO₂^.− generation via a distinct, NOX 2-independent pathway that relied on RyR-mediated mitochondrial Ca²⁺ accumulation. Thus, in NB4 cells, ATO exposure orchestrates a functional crosstalk between discrete Ca²⁺ sources to regulate a cascade of events culminating in NOX 2 activation, mitoO₂^.− production, and initiation of the mitochondrial apoptotic pathway.</p>