Previous studies showed that the activation of Wnt signaling reduced high glucose (HG)-mediated fibroblast damage, but the molecular basis for this phenomenon remains elusive. This study aimed to analyze the level of phosphorylation of GSK3 beta Ser(9) (pGSK3 beta Ser(9)) during HG damage. Moreover, the phosphomimic form of pGSK3 beta Ser(9) was expressed to analyze its effect on cell migration via the phosphorylation of Ikaros. The results revealed that HG treatment significantly reduced the pGSK3 beta Ser(9) level. The overexpression of GSK3 beta Ser(9) D and GSK3 beta Ser(9) A accelerated and inhibited fibroblast cell migration, respectively. P110a knockdown or treatment with SP600125, an inhibitor of JNK, also reduced the pGSK3 beta Ser(9) level under HG condition. Treatment with SP600125 inhibited the migration of fibroblasts, but not in GSK3 beta Ser(9) D-expressing cells. Further, yeast two-hybrid screening and biochemical analysis identified that GSK3 beta interacted and phosphorylated Ikaros at Ser(391). Besides, GSK3 beta Ser(9)D, but not GSK3 beta Ser(9)A, activated Ikaros Ser(391) phosphorylation. Expressing Ikaros or beta-catenin significantly promoted cell migration, suggesting that GSK3 beta modulated cell migration partially via the activation of Ikaros besides beta-catenin signaling under HG condition. The expression of the phosphomimic form of Ikaros Ser(391)D resulted in a significant increase in the extent of cell migration compared with Ikaros under HG condition. Moreover, the Ikaros Ser(391)D DNA-binding affinity toward the ANXA4 promoter increased, and ANXA4 suppression promoted cell migration. In conclusion, the results of this study provided a new regulatory mechanism by which GSK3 beta negatively regulated human skin fibroblast cell migration. (C) 2020 Elsevier Inc. All rights reserved.