Cytochrome b₅₆₁ is a transmembrane hemoprotein unique to neuroendocrine secretory vesicles. It acts as an electron channel and mediates electron transfer from extravesicular ascorbate to intravesicular monodehydroascorbate radical to regenerate ascorbate inside the secretory vesicle for use by copper containing monooxygenases. Two hemes b are allocated on the extravesicular and intravesicular sides of the protein and each has a distinct physiological role, i.e., electron acceptance from ascorbate and electron donation to monodehydroascorbate radical, respectively. Elucidation of the structure and mechanism of redox activity of cytochrome b₅₆₁ and the transmembrane electron transfer between two heme b centers will provide insights into long-range biological electron transfer and may demonstrate paradigm for other emerging members of cytochrome b₅₆₁ family.