Mitochondrial function is critical to maintain high rates of oxidative metabolism supporting energy demands of both spontaneous and evoked neuronal activity in the brain. Mitochondria not only regulate energy metabolism, but also influence neuronal signaling. Regulation of “energy metabolism” and “neuronal signaling” (i.e. neurometabolic coupling), which are coupled rather than independent can be understood through mitochondria’s integrative functions of calcium ion (Ca2+) uptake and cycling. While mitochondrial Ca2+ do not affect hemodynamics directly, neuronal activity changes are mechanistically linked to functional hyperemic responses (i.e. neurovascular coupling). Early in vitro studies lay the foundation of mitochondrial Ca2+ homeostasis and its functional roles within cells. However, recent in vivo approaches indicate mitochondrial Ca2+ homeostasis as maintained by the role of mitochondrial Ca2+ uniporter (mCU) influences system-level brain activity as measured by a variety of techniques. Based on earlier evidence of subcellular cytoplasmic Ca2+ microdomains and cellular bioenergetic states, a mechanistic model of Ca2+ mobilization is presented to understand systems-level neurovascular and neurometabolic coupling. This integrated view from molecular and cellular to the systems level, where mCU plays a major role in mitochondrial and cellular Ca2+ homeostasis, may explain the wide range of activation-induced coupling across neuronal activity, hemodynamic, and metabolic responses.