Fibroblast growth factor 21 (FGF21) is an endocrine FGF that plays a vital role in regulating essential metabolic pathways. FGF21 increases glucose uptake by cells, promotes fatty acid oxidation, reduces blood glucose levels, and alleviates metabolic diseases. However, detailed studies on its stability and biophysical characteristics have not been reported. Herein, we present the overexpression, biophysical characterization, and metabolic activity of a soluble recombinant FGF21 (rFGF21). The far-UV circular dichroism spectra of rFGF21 show a negative trough at 215 nm, indicating that the protein's backbone predominantly adopts a β sheet conformation. rFGF21 shows intrinsic tyrosine fluorescence at 305 nm. Thermal denaturation using differential scanning calorimetry reveals that rFGF21 is relatively thermally unstable, with a melting temperature of 46.8°C (±0.1°C). The urea-induced unfolding of rFGF21 is rapid, with a chemical transition midpoint of 0.4 M. rFGF21 is readily cleaved by trypsin in limited trypsin digestion assays. Isothermal titration calorimetry experiments show that rFGF21 does not bind to heparin. Interestingly, rFGF21 demonstrates proliferative activity in NIH/3T3 fibroblasts and enhances mitochondrial oxidative phosphorylation and fatty acid oxidation in 3T3-L1 adipocytes. These findings provide a crucial framework for the engineering of novel structure-based variants of FGF21 with improved stability and biological activity to treat metabolic disorders.