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Abstract The present study examines the association between the ceremonial use of ayahuasca—a decoction combining the Banistereopsis caapi vine and N,N-Dimethyltryptamine-containing plants—and changes in personality traits as conceived by the Five-Factor model (FFM). We also examine the degree to which demographic characteristics, baseline personality, and acute post-ayahuasca experiences affect personality change. Participants recruited from three ayahuasca healing and spiritual centers in South and Central America (N = 256) completed self-report measures of personality at three timepoints (Baseline, Post, 3-month Follow-up). Informant-report measures of the FFM were also obtained (N = 110). Linear mixed models were used to examine changes in personality and the moderation of those changes by covariates. The most pronounced change was a reduction in Neuroticism dz self-reportT1–T2 =  − 1.00; dz self-reportT1–T3 =  − .85; dz informant-reportT1–T3 =  − .62), reflected in self- and informant-report data. Moderation of personality change by baseline personality, acute experiences, and purgative experiences was also observed.