The structural proximity and functional coupling between the SR (sarcoplasmic reticulum) and mitochondria have been suggested to occur in the heart. However, the molecular architecture involved in the SR-mitochondrial coupling remains unclear. In the present study, we performed various genetic and Ca2+-probing studies to resolve the proteins involved in the coupling process. By using the bacterial 2-hybrid, glutathione transferase pull-down, co-immunoprecipitation and immunocytochemistry assays, we found that RyR2 (ryanodine receptor type 2), which is physically associated with VDAC2 (voltage-dependent anion channel 2), was co-localized in SR-mitochondrial junctions. Furthermore, a fractionation study revealed that VDAC2 was co-localized with RyR2 only in the subsarcolemmal region. VDAC2 knockdown by targeted short hairpin RNA led to an increased diastolic [Ca2+] (calcium concentration) and abolishment of mitochondrial Ca2+ uptake. Collectively, the present study suggests that the coupling of VDAC2 with RyR2 is essential for Ca2+ transfer from the SR to mitochondria in the heart.