CHIR 99021 shows greater than 500-fold selectivity for GSK-3 versus its closest homologs CDC2 and ERK2, as well as other protein kinases. Furthermore, CHIR 99021 shows only weak binding to a panel of 22 pharmacologically relevant receptors and little inhibitory activity against a panel of 23 nonkinase enzymes. CHIR 99021 induces the activation of glycogen synthase (GS) in insulin receptor-expressing CHO-IR cells with EC50 of 0.763 μM. In addition to simulating the actions of insulin, inhibition of GSK-3 by CHIR 99021 (3 μM) increases free cytosolic β-catenin by 1.9-fold, mimicking the canonical Wnt signaling pathway in 3T3-L1 preadipocytes. During any of the first 3 days of differentiation, CHIR 99021 treatment inhibits the preadipocyte differentiation with IC50 of 0.3 μM by blocking induction of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ). Unlike lithium chloride and AR-A014418, CHIR-99021 treatment does not reduce the viability of INS-1E cells even at high concentrations. Instead, CHIR 99021 robustly increases the rate of proliferation of INS-1E cells in a dose-dependent manner, and significantly inhibits INS-E cell death induced by high glucose and high palmitate in a concentration-dependent manner. CHIR 99021 promotes primary beta cell replication in isolated rat islets at concentrations as low as 1 μM, with 2-3 fold increase of cell replication at 5 μM of CHIR 99021 treatment.