Abstract <p>We recently developed a time-correlated single photon counting (TCSPC) spectroscopy approach to investigate the activation mechanisms of the calcium pump SERCA (sarcoplasmic reticulum Ca²⁺-ATPase). Here, we apply this approach to characterize the effects of two chemically distinct SERCA inhibitors, thapsigargin (TG) and cyclopiazonic acid (CPA), and to determine how they differentially modulate SERCA conformational dynamics. Although TG and CPA appear to stabilize similar SERCA states in structural studies, TCSPC reveals fundamentally distinct mechanisms of inhibition. TG stabilizes an inactive conformation that prevents Ca²⁺- and ATP-dependent transitions, effectively trapping SERCA in a nonproductive ‘dead-end’ state. In contrast, CPA attenuates, rather than abolishes, Ca²⁺- and nucleotide-dependent structural transitions, producing graded, concentration-dependent effects that reduce the population of the closed, activation-associated state. Notably, CPA decreases apparent Ca²⁺ affinity only in the presence of a non-hydrolyzable ATP analog, consistent with an ATP-dependent, allosteric mechanism that redistributes conformational populations rather than directly occluding Ca²⁺ binding sites. These findings demonstrate that TCSPC resolves mechanistic differences between inhibitors that appear structurally similar and provide a framework for understanding how distinct allosteric ligands modulate SERCA function.</p> Graphical Abstract <p></p>