Background: Isolated terminal ileitis (ITI) is a phenomenon often observed during colonoscopy, but in most cases, the specific etiology of ITI is unclear. Helicobacter pylori (H. pylori) infection has been reported to be associated with a wide variety of diseases, especially gastrointestinal diseases. Here, we conducted a retrospective study to explore a potential correlation between H. pylori infection and unexplained ITI (UITI). Materials and Methods: The retrospective study was conducted at Sino-French New City Branch of Tongji Hospital, Wuhan, China, from April 2017 to October 2020. All subjects underwent total colonoscopy, with the endoscope being inserted more than 10cm into the terminal ileum. Subjects also received a C-13-urea breath test (C-13-UBT). Data on the age, gender, endoscopic manifestations, and main clinical symptoms of subjects were collected. The presence of H. pylori infection was defined as a positive C-13-UBT result. Logistic regression models were used to analyze the potential correlation between H. pylori infection and UITI. Results: There were 247 subjects (25.1%) in the H. pylori (+) group and 739 subjects (74.9%) in the H. pylori (-) group. The prevalence of UITI in the H. pylori (+) group was significantly lower than that in the H. pylori (-) group (OR = 0.518; 95% CI 0.281-0.956; P = 0.035), and there was no difference in other clinical features between groups. Stratification analysis results showed that there was an inverse association between H. pylori infection and UITI in subjects with age <60 (P = 0.046). Conclusion: These data showed that H. pylori infection was negatively correlated with UITI. Additional studies are needed to validate these findings in a larger cohort as well as to explore the underlying mechanisms.