Axin is a recently identified protein encoded by the fused locus in mice that is required for normal vertebrate axis formation. We have defined a 25-amino-acid sequence in axin that comprises the glycogen synthase kinase 3β (GSK-3β) interaction domain (GID). In contrast to full-length axin, which has been shown to antagonize Wnt signaling, the GID inhibits GSK-3β in vivo and activates Wnt signaling. Similarly, mutants of axin lacking key regulatory domains such as the RGS domain, which is required for interaction with the adenomatous polyposis coli protein, bind and inhibit GSK-3β in vivo, suggesting that these domains are critical for proper regulation of GSK-3β activity. We have identified a novel self-interaction domain in axin and have shown that formation of an axin regulatory complex in vivo is critical for axis formation and GSK-3β activity. Based on these data, we propose that the axin complex may directly regulate GSK-3β enzymatic activity in vivo. These observations also demonstrate that alternative inhibitors of GSK-3β can mimic the effect of lithium in developing Xenopus embryos.