The nephron is composed of distinct segments that perform unique physiological functions. Little is known about how multipotent nephron progenitor cells differentiate into different nephron segments. It is well known that β-catenin signaling regulates the maintenance and commitment of mesenchymal nephron progenitors during kidney development. However, it is not fully understood how it regulates nephron segmentation after nephron progenitors undergo mesenchymal-to-epithelial transition. To address this, we performed β-catenin loss-of-function and gain-of-function studies in epithelial nephron progenitors in the mouse kidney. Consistent with a previous report, the formation of the renal corpuscle was defective in the absence of β-catenin. Interestingly, we found that epithelial nephron progenitors lacking β-catenin were able to form presumptive proximal tubules but that they failed to further develop into differentiated proximal tubules, suggesting that β-catenin signaling plays a critical role in proximal tubule development. We also found that epithelial nephron progenitors lacking β-catenin failed to form the distal tubules. Expression of a stable form of β-catenin in epithelial nephron progenitors blocked the proper formation of all nephron segments, suggesting tight regulation of β-catenin signaling during nephron segmentation. This work shows that β-catenin regulates the formation of multiple nephron segments along the proximo-distal axis of the mammalian nephron.