The mitochondrial carrier family (solute carrier family 25) is a super family of nuclearencoded transporters localized on the inner mitochondrial membranes. In human, the mitochondrial carrier family has 53 members, all with a ternary structure of six transmembrane α-helices. The structure of mitochondrial carrier family has three repeats of conservative motifs. The members of this family connect the inter membrane space and matrix of mitochondria, and transport various important small molecules across the inner membrane. In the present review, we have highlighted the limitations of traditional research methods to gain the accurate knowledge of membrane proteins. We have focused on recent emerging computational strategies such as molecular modeling, molecular dynamics, and quantitative structure–activity relationship to predict the structure and function of membrane proteins at atomic resolution in the absence of experimental data. This review aims to summarize a comprehensive introduction of recent discoveries about the biological investigations of the structure and transport mechanism of mitochondrial carriers, which are useful for further investigation on diseases and drug developments related to mitochondrial carrier deficiency, especially the combination of traditional experiments and emerging computational strategies.