The role of hyperglycemia in diabetic changes of the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) has not been clearly established. We therefore determined whether glucose modulates IGFBP synthesis and stability in vitro. Porcine vascular smooth muscle cells (pSMC) cultured in low glucose (pSMC-L) had 2.1-fold more IGFBP-4 in the conditioned medium compared with pSMC cultured in high glucose (pSMC-H) (P < 0.01). In contrast, IGFBP-2 levels remained constant. Although pSMC-H and pSMC-L cultures expressed similar levels of IGFBP-4 messenger RNA, in vitro protease assays demonstrated an increase in IGFBP-4 proteolysis in pSMC-H conditioned medium compared with pSMC-L conditioned medium (P < 0.01). The protease had properties similar to a previously characterized IGFBP-4 protease. The addition of 20 mM mannitol to pSMC-L cultures did not decrease IGFBP-4 levels, suggesting that the difference in IGFBP-4 proteolysis was not osmotically induced. The change was not due to selection bias, because cultures that were initially isolated from aortic explants in high and low glucose still exhibited the glucose-dependent difference in IGFBP-4 proteolytic activity. The results suggest that high glucose acts on pSMC to induce a change in IGFBP-4 proteolytic activity, which results in increased IGF-I availability to its receptors thereby enhancing the SMC proliferative response.