Glucose homeostasis was examined in male transgenic (Tg) mice that overexpressed the human insulin-like growth factor (IGF)-binding protein (IGFBP)-3 cDNA, driven by either the cytomegalovirus (CMV) or the phosphoglycerate kinase (PGK) promoter. The Tg mice of both lineages demonstrated increased serum levels of human (h) IGFBP-3 and total IGF-I compared with wild-type (Wt) mice. Fasting blood glucose levels were significantly elevated in 8-wk-old CMV-binding protein (CMVBP)-3- and PGK binding protein (PGKBP)-3-Tg mice compared with Wt mice: 6.35 +/- 0.22 and 5.22 +/- 0.39 vs. 3.99 +/- 0.26 mmol/l, respectively. Plasma insulin was significantly elevated only in CMVBP-3-Tg mice. The responses to a glucose challenge were significantly increased in both Tg strains: area under the glucose curve = 1,824 +/- 65 and 1,910 +/- 115 vs. 1,590 +/- 67 mmol. l(-1). min for CMVBP-3, PGKBP-3, and Wt mice, respectively. The hypoglycemic effects of insulin and IGF-I were significantly attenuated in Tg mice compared with Wt mice. There were no differences in adipose tissue resistin, retinoid X receptor-alpha, or peroxisome proliferator-activated receptor-gamma mRNA levels between Tg and Wt mice. Uptake of 2-deoxyglucose was reduced in muscle and adipose tissue from Tg mice compared with Wt mice. These data demonstrate that overexpression of hIGFBP-3 results in fasting hyperglycemia, impaired glucose tolerance, and insulin resistance.