The adenomatous polyposis coli (APC) protein has a tumor-suppressor function by acting as a negative regulator of the Wnt signaling pathway. While its role as a tumor suppressor is well-defined, the post-translational modifications that regulate APC stability are not fully understood. Here we showed that MKRN1, an E3 ligase, could directly interact with and ubiquitylate APC, promoting its proteasomal degradation. In contrast, an E3 ligase-defective MKRN1 mutant was no longer capable of regulating APC, indicating that its E3 ligase activity is required for APC regulation by MKRN1. Strengthening these results, MKRN1 ablation resulted in reduced β-catenin activity and decreased expression of Wnt target genes. The ability of the Wnt-dependent pathway to induce cancer cell proliferation, migration, and invasion was impaired by MKRN1 depletion, but restored by simultaneous APC knockdown. Taken together, these results demonstrate that MKRN1 functions as a novel E3 ligase of APC that positively regulates Wnt/β-catenin-mediated biological processes.