Objective: To assess the effect of azithromycin mass drug administration regimens on spleen rates in children aged 0-5 years. Methods: Clinical assessment of spleen size was carried out during a cluster-randomised trial of azithromycin mass treatment for trachoma elimination in The Gambia. Twenty-four communities received three annual mass treatments with azithromycin, and 24 communities received treatment at baseline only. Results: At the 30-month follow-up, 3646 children aged 0-5 years had spleen examination and measurement. Palpable splenomegaly was significantly lower in annually treated vs. baseline-only treatment communities and in treated vs. untreated children at 24 months in the annual treatment arm. Conclusion: The results suggest an effect of azithromycin on spleen rates at the individual level and are most plausibly due to the antimalarial effects of azithromycin.