Prostaglandin E2 receptor 4 (EP4) is one of the receptors for Prostaglandin E2 and plays important roles in various biological functions. EP4 antagonists have been used as an anti-inflammatory drug. To investigate the effects of an EP4 antagonist on the endogenous metabolism in a holistic manner, we employed a mouse model, and obtained metabolic and transcriptomic profiles of multiple biological matrices, including serum, liver and urine of mice with and without EP4 antagonist (L-161982) exposure. We showed that the EP4 antagonist caused significant changes in fatty acid metabolism, choline metabolism and nucleotide metabolism. EP4 antagonist exposure also induced oxidative stress to mice. Our research is the first of its kind to report the information on the alteration of metabolism associated with an EP4 antagonist. This information could further our understanding of current and new biological functions of EP4.