Allicin, one of the main bioactive compounds in garlic, is an excellent feed additive. It is unknown whether allicin affects the expression of P-gp and BCRP, 2 important ABC efflux transporters related to the pharmacokinetics of antimicrobials in chickens. In this study, by bidirectional transport test and broiler jejunum in situ perfusion, we found that allicin inhibited the efflux transport of P-gp and BCRP for their substrates sulfadiazine and florfenicol, 2 commonly used antimicrobials in broilers. Furthermore, we observed that allicin decreased the mRNA expression of chicken jejunum P-gp and BCRP. Pretreatment with allicin changed the pharmacokinetic behavior of orally administered sulfadiazine, by increasing AUC (41.85 vs. 31.24, P < 0.01) and Cmax values (9.82 vs. 8.40, P < 0.05) and decreasing CLZ (0.45 vs. 0.62, P < 0.01). Similarly, pretreatment with allicin also altered pharmacokinetics of orally administered florfenicol, by increasing AUC (18.38 vs. 13.52, P < 0.01) and decreasing CLZ. The results indicate that allicin could inhibit jejunum P-gp and BCRP expression and efflux function, thereby increasing the plasma concentrations of their substrates in broilers.