Circulating levels of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs), are altered in human obesity and may contribute to its pathology. TIMP-2 exerts MMP-dependent (MMP inhibition and pro-MMP-2 activation) and MMP-independent functions. To assess the role of TIMP-2 in a murine model of nutritionally induced obesity, weight gain in wild-type and TIMP-2 deficient [knockout (KO)] mice fed a chow or high-fat diet (HFD) was determined. The effects of diet on glucose tolerance and insulin sensitivity, as well as pancreatic β-cell and adipocyte physiology, were assessed. Chow-fed TIMP-2 KO mice of both sexes became obese but maintained relatively normal glucose tolerance and insulin sensitivity. Obesity was exacerbated on the HFD. However, HFD-fed male, but not female, TIMP-2 KO mice developed insulin resistance with reduced glucose transporter 2 and pancreatic and duodenal homeobox 1 levels, despite increased β-cell mass and hyperplasia. Thus, although β-cell mass was increased, HFD-fed male TIMP-2 KO mice develop diabetes likely due to β-cell exhaustion and failure. TIMP-2 mRNA, whose expression was greatest in sc adipose tissue, was down-regulated in HFD-fed wild-type males, but not females. Furthermore, HFD increased membrane type 1-MMP (MMP-14) expression and activity in male, but not female, sc adipose tissue. Strikingly, MMP-14 expression increased to a greater extent in TIMP-2 KO males and was associated with decreased adipocyte collagen. Taken together, these findings demonstrate a role for TIMP-2 in maintaining extracellular matrix integrity necessary for normal β-cell and adipocyte physiology and that loss of extracellular matrix integrity may underlie diabetic and obesogenic phenotypes.