Objective: The purpose of the study was to non-invasively characterize and discriminate brown adipose tissue (BAT) from white adipose tissue (WAT) in rats using spectral computed tomography (CT) with histological validation. Materials and Methods: A lipid-containing phantom (lipid fractions from 0% to 100%) was imaged with spectral CT. An in vivo, non-enhanced spectral CT scan was performed on 24 rats, and fat concentrations of BAT and WAT were measured. The rats were randomized to receive intraperitoneal treatment with norepinephrine (NE) (n = 12) or saline (n = 12). Non-enhanced and enhanced spectral CT scans were performed after treatment to measure the elevation of iodine in BAT and WAT. The BAT/ aorta and WAT/aorta ratios were calculated and compared, after which isolated BAT and WAT samples were subjected to histological and uncoupling protein 1 (UCP1) analyses. Results: The ex-vivo phantom study showed excellent linear fit between measured fat concentration and the known gravimetric reference standard (r2 = 0.996). In vivo, BAT had significantly lower fat concentration than WAT (p < 0.001). Compared to the saline group, the iodine concentration of BAT increased significantly (p < 0.001) after injection of NE, while the iodine concentration of WAT only changed slightly. The BAT/aorta ratio also increased significantly after exposure to NE compared to the saline group (p < 0.001). Histological and UCP1 expression analyses supported the spectral CT imaging results. Conclusion: The study consolidates spectral CT as a new approach for non-invasive imaging of BAT and WAT. Quantitative analyses of BAT and WAT by spectral CT revealed different characteristics and pharmacologic activations in the two types of adipose tissue.