Objective: The aim of this study was to determine whether the presence of Helicobacter pylori (H. pylori) infection and multidrug resistance protein 1 (MDR1) C3435T polymorphism had an influence on P-glycoprotein (P-gp) expression in the upper gastrointestinal tract. Methods: A total of 76 patients who underwent upper gastroendoscopy at Sir Charles Gairdner Hospital in Western Australia from October 2010 to July 2011 were enrolled in the study. Antral and duodenal biopsies were collected for P-gp examination. Blood samples were taken and analyzed for MDR1 C3435T polymorphism. H.pylori infection status was confirmed by culture and polymerase chain reaction. Results: A significant difference was found in P-gp expression between H.pylori-positive and H.pylori-negative patients (P=0.028). For the MDR1 C3435T polymorphism, the TT genotype had a significantly lower P-gp expression compared with the CC genotype in antral specimens (P=0.041). The homozygous TT genotype with H.pylori infection was also significantly different in P-gp expression compared with H.pylori-negative patients (P=0.029). Conclusions: P-gp expression in the upper gastrointestinal tract is associated with H.pylori infection, and the TT genotype appeared to be associated with lower P-gp expression than the CC genotype in the stomach.