Objectives <p>We aimed to evaluate the safety and efficacy of cotrimoxazole for preventing infections in patients with glomerulonephritis (GN) receiving rituximab (RTX) therapy.</p> Study design and methods <p>This single-center, open-label randomized controlled trial enrolled 150 patients with GN who received RTX at Xijing Hospital between October 2022 and December 2023. Participants were randomized 1:1 to either the cotrimoxazole prophylaxis group (<i>n</i> = 75) or the non-cotrimoxazole group (<i>n</i> = 75). The primary endpoint was the cumulative incidence of infection over a 180-day follow-up period. Secondary endpoints included the incidence of severe infections and the rate of adverse events (AEs) attributed to cotrimoxazole.</p> Intervention <p>All patients received RTX at a dose of 1000 mg intravenously on days 1 and 14. CD19 + B-cell counts were monitored monthly for 6-month post-initial infusion; if CD19 + B cells remained detectable at the 6-month mark, an additional 1000-mg RTX dose was administered.</p> Cotrimoxazole prophylaxis protocol <p>Patients in the intervention group initiated oral cotrimoxazole on the same day as their first RTX infusion, with one tablet daily for seven consecutive days. Prophylaxis was repeated for 7 days following each subsequent RTX infusion, resulting in a total of 14 days of prophylaxis over the study period.</p> The non-cotrimoxazole group <p>They received no cotrimoxazole prophylaxis.</p> Results <p>Over 180&#xa0;days of follow-up, 8 infectious episodes occurred in 7 patients in the cotrimoxazole group, compared with 19 episodes in 16 patients in the control group. The annualized incidence density of infections (per 100 person-years) was 20.80 (95% <i>CI</i> 11.36–30.24) in the cotrimoxazole group versus 54.83 (95% <i>CI</i> 43.09–66.57) in the control group. The hazard ratio (HR) for the cumulative incidence of infection was 0.39 (95% <i>CI</i> 0.17–0.88; log-rank test, <i>P</i> = 0.029), indicating a significant reduction in infection risk with cotrimoxazole prophylaxis.</p> Conclusions <p>Our findings demonstrate that cotrimoxazole prophylaxis for 1-week post-RTX infusion effectively prevents post-RTX infectious complications, reduces infection-related mortality, and does not elevate the risk of serious AEs. However, it should be noted that this study was a single-center investigation without a placebo control.</p> Trial registration <p>The study was registered on the Chinese Clinical Trial Registry (trial registration identifier: ChiCTR2200063564).</p>