Gallstones disease is classified as cholesterol, pigment and mixed gallstones. Pigment gallstones is a common disease, and some patients experienced complications such as biliary pain and cholecystitis. Defatted walnut powder extract (DWPE) is extracted from the main by-product after oil extraction of the functional food walnut and can treat cholesterol gallstones. However, the effect of DWPE on pigment gallstones has not been reported. Thus, this paper aimed at exploring the potential mechanism of alleviating pigment gallstones and the metabolism of DWPE in vitro by UPLC-Q-Exactive Orbitrap MS to elucidate potential pharmacodynamic substances. It was found that DWPE significantly reduced the incidence of gallstones, the contents of unconjugated bilirubin and calcium, the inflammation of gallbladder tissue, the edema and congestion of gallbladder tissue, and the infiltration of inflammatory cells. In metabolism, a total of 35 metabolites of DWPE, including its main compounds ellagic acid and glansreginin A, were characterized in the intestine with various metabolic reactions. Presumably, the small molecular compounds generated after the metabolism by intestinal bacteria may be the pharmacodynamic substances of DWPE on pigment gallstones. This study revealed the effect of DWPE in treating pigment gallstones and their metabolism in vitro.