Background: The prevalence of self-reported gastrointestinal (GI) symptoms and distress is high, but few studies have quantified their impact on health-related quality of life (HRQoL). Methods: We conducted a prospective cohort study of patients with HIV in care in Ontario, Canada (2007-2014). General linear mixed models were used to assess the impact of GI symptoms (diarrhea/soft stool, nausea/vomiting, bloating/painful abdomen, loss of appetite, weight loss/wasting) and distress (range: 0-4) on physical and mental HRQoL summary scores (range: 0-100) measured by the Medical Outcomes Survey SF-36. Results: A total of 1787 participants completed one or more questionnaires {median 3 [interquartile range (IQR): 1-4]}. At baseline, 59.0% were men who had sex with men, 53.7% white, median age 45 (IQR: 38-52), median CD4(+) count 457 (IQR: 315-622), and 71.0% had undetectable HIV viremia. The mean (standard deviation [SD]) mental and physical HRQoL scores were 49.2 (8.6) and 45.3 (13.0), respectively. In adjusted models, compared with those reporting no symptoms, all GI symptom distress scores from 2 ("have symptom, bothers me a little") to 4 ("have symptom, bothers a lot") were associated with lower mental HRQoL. Loss of appetite distress scores >= 1; scores >= 2 for diarrhea, nausea/vomiting, and bloating; and a score >= 3 for weight loss were independently associated with lower physical HRQoL scores (P < 0.0001). Increasing GI symptom distress is associated with impaired mental and physical HRQoL (P < 0.0001). Conclusions: Increasing GI symptom distress is associated with impaired mental and physical HRQoL. Identifying, treating, and preventing GI symptoms may reduce overall symptom burden and improve HRQoL for patients with HIV.