Wnt signaling regulates cell fate determination, proliferation, and survival, among other processes. Certain Wnt ligands stabilize the -catenin protein, leading to -catenin’s ability to activate T cell factor (TCF)-regulated genes. In the absence of Wnts, -catenin is phosphorylated at defined serine and threonine residues in its amino (N)-terminus. The phosphorylated -catenin is recognized by a -transducin repeat-containing protein (TrCP) and associated ubiquitin ligase components. The serine/threonine residues and TrCP-binding site in -catenin’s N-terminal region constitute a key regulatory motif targeted by somatic mutations in human cancers, resulting in constitutive stabilization of the mutant -catenin proteins. Structural studies have implicated -catenin lysine 19 (K19) as the major target for TrCP-dependent ubiquitination, but K19 mutations in cancer have not been reported. We studied the consequences of single amino acid substitutions of the only two lysine (K) residues in -catenin’s N-terminal 130 amino acids. Mutation of K19 minimally affected -catenin’s levels and functional activity, and mutation of K49 led to reduced -catenin levels and function. In contrast, -catenin proteins with substitutions at both K19 and K49 positions were present at elevated levels and had the ability to potently activate TCF transcription and promote neoplastic transformation. We furthermore demonstrate that the K19/K49 double mutant forms of -catenin are stabilized as a result of reduced TrCP-dependent ubiquitination. Our findings suggest that K19 is a primary in vivo site of TrCP-dependent ubiquitination, and K49 may be a secondary or cryptic site. Moreover, our results inform understanding of why single amino acid substitutions at lysine 19 or 49 have not been reported in human cancer.