Obesity is associated with a chronic low grade inflammation characterized by macrophage infiltration of adipose tissue (AT) that may underlie development of insulin resistance and type 2 diabetes. Osteopontin (OPN) is a multifunctional protein involved in various inflammatory processes, cell migration, and tissue remodeling. Since these processes occur in AT of obese patients, we studied in detail the regulation of OPN expression in human and murine obesity. The study included 20 morbidly obese patients and 20 age- and sex-matched control subjects, as well as two models (diet-induced and genetic) of murine obesity. In high-fat diet-induced and genetically obese mice, OPN expression was drastically upregulated in AT (40 and 80-fold, respectively), but remained largely unaltered in liver (< 2-fold). Moreover, OPN plasma concentrations remained unchanged in both murine models of obesity, suggesting a particular local but not systemic importance for OPN. OPN expression was strongly elevated also in AT of obese patients compared to lean subjects in both omental and subcutaneous AT. In addition, we detected three OPN isoforms to be expressed in human AT and, strikingly, an obesity-induced alteration of the OPN isoform expression pattern. Analysis of AT cellular fractions revealed that OPN is exceptionally highly expressed in AT macrophages (ATMs) in humans and mice. Moreover, OPN expression in ATMs was strongly upregulated by obesity. In conclusion, our data point towards a specific local role of OPN in obese AT. Hence, OPN could be a critical regulator in obesity-induced AT inflammation and insulin resistance.