Protein reabsorption in renal proximal tubules is essential for maintaining nutrient homeostasis. Renal proximal tubule-specific gene knockout is a powerful method to assess the function of genes involved in renal proximal tubule protein reabsorption. However, the lack of inducible renal proximal tubule-specific Cre recombinase-expressing mouse strains hinders the study of gene function in renal proximal tubules. To facilitate the functional study of genes in renal proximal tubules, we developed anAMNCreERT2knock-in mouse strain expressing a Cre recombinase–estrogen receptor fusion protein under the control of the promoter of theamnionless (AMN)gene, a protein reabsorption receptor in renal proximal tubules.AMNCreERT2knock-in mice were generated using the CRISPR/Cas9 strategy, and the tissue specificity of Cre activity was investigated using the Cre/loxP reporter system. We showed that the expression pattern of CreERT2-mEGFP inAMNCreERT2mice was consistent with that of the endogenousAMNgene. Furthermore, we showed that the Cre activity inAMNCreERT2knock-in mice was only detected in renal proximal tubules with high tamoxifen induction efficiency. As a proof-of-principle study, we demonstrated that renal proximal tubule-specific knockout ofExoc4using AMNCreERT2led to albumin accumulation in renal proximal tubular epithelial cells. TheAMNCreERT2mouse is a powerful tool for conditional gene knockout in renal proximal tubules and should offer useful insight into the physiological function of genes expressed in renal proximal tubules.