Mitochondrial calcium uptake plays a critical role in various cellular functions. After half a century of extensive studies, the molecular components and important regulators of the mitochondrial calcium uptake complex have been identified. However, the mechanism by which these protein molecules interact with one another and coordinate to regulate calcium passage through mitochondrial membranes remains elusive. Here, we summarize recent progress in the structural and functional characterization of these important protein molecules, which are involved in mitochondrial calcium uptake. In particular, we focus on the current understanding of the molecular mechanism underlying calcium through two mitochondrial membranes. Additionally, we provide a new perspective for future directions in investigation and molecular intervention.