Gallstone disease (GD) has been associated with low serum levels of plant sterols. We evaluated the impact of laparoscopic Roux-en-Y gastric bypass (LRYGB) and non-alcoholic fatty liver disease (NAFLD) on the association of GD with low levels of serum plant sterols. Two hundred forty-two consecutive morbidly obese patients were recruited to this prospective study. Histological analysis of liver biopsy to diagnose NAFLD was performed. Bile sample was taken during the LRYGB. Associations of GD with serum non-cholesterol sterol to cholesterol ratios, measured using gas liquid chromatography and with mRNA expression of genes participating in the cholesterol, bile, and fatty acid metabolism in the liver, were analyzed. Out of the 242 participants, 95 had GD. Lower weight (p = 0.002) and female sex (p = 0.0006) were associated with GD. Serum plant sterols, campesterol (p = 0.003), sitosterol (p = 0.002), and avenasterol (p = 0.015), were lower in patients with GD compared to those without GD. This association remained significant after adjustment for NAFLD, use of statin medication, and previous laparoscopic cholecystectomy (LCC). Levels of sitosterol (p = 0.001) and campesterol (p = 0.001) remained lower in obese individuals with GD also after obesity surgery. Liver mRNA expression of genes regulating cholesterol synthesis and bile metabolism was increased in individuals with GD. Serum plant sterols were lower in patients with GD independent of NAFLD, history of LCC, use of statin medication, and weight loss after LRYGB. Low serum plant sterols in patients with GD suggest potentially inherited alterations in sterol absorption and biliary transport in subjects susceptible for GD.