The kidney are the major organs determining the drug elimination, and membrane transporters play a central role. There are reports that there are sex differences in the expression levels of transporters that are important in drug elimination function. However, the mechanism of sex difference formation is unclear. The one of reason is that the low abundance and hydrophobic features of membrane proteins, such as transporters, make it difficult to analyze the proteins. Therefore, we developed an original membrane proteomics method to analyze membrane proteins comprehensively with high sensitivity. In this study, we used this method to comprehensively analyze the membrane protein expression level of the Four Core Genotypes (FCG) model, in which the Sry gene was moved from the Y-chromosome to an autosome, allowing distinction between gonadal and sex chromosome effects. Membrane proteomics was performed on brush border membrane vesicles from FCG mice and obtained a membrane protein profile according to gonad and sex chromosomes. Next, bioinformatics analyses were conducted to identify proteins and biological pathways that are important for gonad-derived and sex chromosome-derived sex differentiation. It is expected that this result will be an important information on sex differences of drug elimination in the kidney.