Coccidiosis is a major intestinal disease affecting broiler chickens. Tributyrin (TB) is a valid alternative to butyrate acid, which was associated with the improvement of performance and attenuation of intestinal inflammation in animal production. However, there are few reports on TB as a prophylactic treatment against coccidiosis in broilers. The aim of the study was to investigate the effects of TB supplementation on performance and intestinal health of broiler chickens post coccidiosis vaccination with a mixed-species Eimeria. In the first experiment, 612 broiler chicks were randomly assigned to two treatments with six replicates. Treatments included no TB supplementation and coccidiosis vaccination (CV1), or TB supplementation (400 mg/kg) and coccidiosis vaccination (TBCV1). On day 5, all broilers received a single vaccine dose. Performance, intestinal histopathology, clinical severity, and fecal oocyst counts were evaluated from day 1 to day 63. TB supplementation resulted in a nonsignificant effect on body weight gain (BWG), feed intake (FI), and mortality-corrected feed conversion ratio (FCR), except in increased FI on days 22–42 (P < 0.05). The TBCV1 group had increased (P < 0.05) villi heights in the duodenum and increased (P < 0.05) villi widths in the ileum on day 63 of age and reduced oocyst shedding on days 19–26 compared to CV1(P < 0.05). The route of administration in the second experiment was different from the first experiment in which the seeder birds (half of birds from each pen) received a tenfold dose on day 5. TB supplementation in broilers resulted in increased (P < 0.05) BWG and reduced (P < 0.05) FCR on days 22–42, and increased (P < 0.05) villi heights in the duodenum and increased (P < 0.05) villi widths in the ileum on day 63 of age, as well as a lower frequency (P < 0.05) of intestinal hemorrhage on days 13–62 and reduced (P < 0.001) oocyst shedding on day 5 post-Eimeria challenge. In conclusion, the study demonstrated that TB can be considered as a feed additive for protecting broilers from coccidiosis on days 22–42.