AbstractBackground:  Wnt signalling plays a critical role in many developmental processes and tumorigenesis. Wnt/β‐catenin signalling induces the stabilization of cytosolic β‐catenin, which interacts with TCF/LEF‐1 transcription factors, thereby inducing expression of Wnt‐target genes. Recent evidence suggests that a specific MAP kinase pathway involving the MAP kinase kinase kinase TAK1 and the MAP kinase NLK counteract Wnt signalling.Results:  To identify NLK‐interacting proteins, we performed yeast two‐hybrid screening. We isolated the gene HMG2L1 and showed that injection of Xenopus HMG2L1 (xHMG2L1) mRNA into Xenopus embryos inhibited Wnt/β‐catenin‐induced axis duplication and expression of Wnt/β‐catenin target genes. Moreover, xHMG2L1 inhibited β‐catenin‐stimulated transcriptional activity in mammalian cells.Conclusions:  Our findings indicate that xHMG2L1 may negatively regulate Wnt/β‐catenin signalling, and that xHMG2L1 may play a role in early Xenopus development together with NLK.