Wnt/b-catenin (CTNNB1) signaling is crucial for the proliferation and maintenance of intestinal stem cells (ISC), but excessive activation leads to ISC expansion and eventually colorectal cancer. Thus, negative regulators are required to maintain optimal levels of Wnt/b-catenin signaling. Aminoacyl-tRNA synthetase-interacting multifunctional proteins (AIMP) function in protein synthesis, but have also been implicated in signaling cascades affecting angiogenesis, immunity, and apoptosis. In this study, we investigated the relationship between AIMP2 and Wnt/b-catenin signaling in a murine model of intestinal homeostasis and tumorigenesis. Hemizygous deletion of Aimp2 resulted in enhanced Wnt/b-catenin signaling, increased proliferation of cryptic epithelial cells, and expansion of ISC compartments. In an Apc Min/þ background, Aimp2 hemizygosity increased adenoma formation. Mechanistically, AIMP2 disrupted the interaction between AXIN and Dishevelled-1 (DVL1) to inhibit Wnt/b-catenin signaling by competing with AXIN. Furthermore, AIMP2 inhibited intestinal organoid formation and growth by suppressing Wnt/b-catenin signaling in an Aimp2 gene dosage-dependent manner. Collectively, our results showed that AIMP2 acts as a haploinsufficient tumor suppressor that fine-tunes Wnt/b-catenin signaling in the intestine, illuminating the regulation of ISC abundance and activity. Cancer Res; 76(15); 4559-68. Ó2016 AACR.