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Background: Although humans and animals are often simultaneously exposed to a variety of mycotoxins via feed and food consumption, in which aflatoxin B1 (AFB1) and deoxynivalenol (DON) is one of the most prevalent combination of mycotoxins. Many toxicological studies have merely focused on the impacts of single mycotoxins. In the present study, the combined toxicity and the related mechanism of AFB1 and DON to zebrafish (Danio rerio) were investigated. Results: Results exhibited that DON had lower toxicity to embryonic zebrafish with a 7-day LC50 of 218.3 mg a.i. L−1 in comparison to AFB1 (0.031 mg a.i. L−1). The mixture of AFB1 and DON elicited an additive combined effect on zebrafish embryos. The levels of CAT, caspase-3, and T4 markedly varied in most single and mixture groups. The expressions of four genes (cas3, apaf-1, cc-chem, and cyp19a) associated with oxidative stress, cellular apoptosis, immune system, and endocrine system were markedly varied upon the mixture exposure in comparison to the corresponding single exposure of AFB1 or DON. Conclusions: Our results revealed that the impacts of a mixture could not be estimated solely based on toxicities of the single mycotoxins. Taken together, our comprehensive investigation on the mycotoxin mixtures and their potential mechanisms could better reflect the reality of mycotoxin contamination in food and feed. [ABSTRACT FROM AUTHOR]