Fibroblast growth factor (FGF) 21 is a class of hepatokines that plays a protective role against obesity, insulin resistance, and liver damage. Despite this, protective effects of FGF21 in human appear to be minimal, possibly due to its proteolytic cleavage by the fibroblast activation protein (FAP). Here, we presented a novel FAP inhibitor, BR103354, and described its pharmacological activities as a potential therapeutic agent for the treatment of metabolic disorders. BR103354 inhibited FAP with an IC50 value of 14 nM, showing high selectivity against dipeptidyl peptidase (DPP)-related enzymes and prolyl oligopeptidase (PREP). In differentiated 3T3/L1 adipocytes, the addition of FAP diminished hFGF21-induced Glut1 and phosphorylated levels of ERK, which were restored by BR103354. BR103354 exhibited good pharmacokinetic properties as evidenced by oral bioavailability of 48.4% and minimal hERG inhibition. Single co-administration of BR103354 with hFGF21 reduced nonfasting blood glucose concentrations, in association with increased intact form of hFGF21 in ob/ob mice. Additionally, chronic treatment of BR103354 for 4 weeks reduced nonfasting blood glucose concentrations with improved glucose tolerance and with reduced triglyceride (TG) content in liver of ob/ob mice. Consistently, BR103354 improved hepatic steatosis and fibrosis in a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-induced non-alcoholic steatohepatitis (NASH) mouse model. FAP inhibitory effects of BR103354 were confirmed in normal cynomolgus monkeys. Together, BR103354 acts as an effective FAP inhibitor in vitro and in vivo, thereby demonstrating its potential application as an anti-diabetic and anti-NASH agent.