The inositol 1,4,5-trisphosphate receptors (IP₃Rs) form clusters following agonist stimulation, but its mechanism remains controversial. In this study, we visualized the clustering of green fluorescent protein (GFP)-tagged type 3 IP₃R (GFP-IP₃R3) in cultured living cells using confocal microscopy. Stimulation with ATP evoked GFP-IP₃R3 clustering not only in cells with replete Ca²⁺-stores but also in cells with depleted Ca²⁺ stores. Thapsigargin (ThG) and ionomycin failed to mimic the ATP-induced cluster formation despite the continuous elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Application of IP₃ caused GFP-IP₃R3 clustering in permeabilized cells, and the response was completely inhibited by heparin, a competitive inhibitor of IP₃R. Experiments using LIBRAv, an IP₃ biosensor, showed that ATP significantly stimulated IP₃ generation even in store-depleted cells. We also found that pretreatment with ThG accelerated or enhanced the ATP-induced clustering in both the presence and absence of extracellular Ca²⁺. When permeabilized cells were stimulated with the threshold of IP₃, the GFP-IP₃R3 clustering clearly occurred in Ca²⁺-free medium but not in Ca²⁺-containing medium. These results strongly support the hypothesis that the agonist-induced clustering of IP₃R is triggered by IP₃ binding, rather than [Ca²⁺]_i elevation. Although depletion of the Ca²⁺ store by itself does not cause the clustering, it may increase the sensitivity of IP₃R to cluster formation, leading to facilitation of IP₃-triggered clustering.