Rotavirus (RV) is one of the main pathogens that induce infantile diarrhea and by now no effective drugs are available for RV-induced infantile diarrhea. Thus the development of novel models is of vital importance for the pathological research of RV-induced infantile diarrhea, as well as the progress of the associated treatment strategy. Here we introduced for the first time that RV-Wa strain and RV-SA-11 strain could infect 5 dpf(day post fertilization) and 28 dpf larvae, to induce infantile diarrhea model that was highly consistent with the clinical infection of infants. RV infection significantly changed the signs, survival rate and inflammation of larvae. Some important indicators, including the levels of RV antigen VP4 and VP6, the in vivo RV tracking, and the RV particles were also analyzed, which collectively demonstrated that the model was successfully established. More importantly, we also determined the potentials of the proposed RV-infected zebrafish model for anti-viral drug assessment. In conclusion, we established a RV-infected zebrafish model with formulated relevant indicators both larvae and adult fish, which might be served as a high throughput platform for antiviral drug screening. • Our research found that rotavirus SA-11 and WA strains with a certain virus titer could infect 5dpf and 28dpf zebrafish larvae, but could not infect 90dpf zebrafish. • After rotavirus infection with 28dpf zebrafish larvae, obvious diarrhea, anal swelling and other symptoms occurred, and the diarrhea lasted for 2–3 days, which was similar to RV infection in suckling mouse model. • The anti-RV effect of Pinocembrin in RV infected zebrafish model was consistent with classical Caco2 cell model, and zebrafish model could be used for anti-RV drug screening with the advantage of high throughput and high content. [ABSTRACT FROM AUTHOR]