Rotavirus (RV) is the major causes of severe diarrhea in infants and young children under five years of age. There are no effective drugs for the treatment of rotavirus in addition to preventive live attenuated vaccine. Recent evidence demonstrates that microRNAs (miRNAs) can affect RNA virus replication. However, the antiviral effect of miRNAs during rotavirus replication are largely unknown. Here, we determined that miR-7 is upregulated during RV replication and that it targets the RV NSP5 (Nonstructural protein 5). Results suggested that miR-7 affected viroplasm formation and inhibited RV replication by down-regulating RV NSP5 expression. Up-regulation of miR-7 expression is a common regulation method of different G-type RV-infected host cells. Then, we further revealed the antiviral effect of miR-7 in diarrhea suckling mice model. MiR-7 is able to inhibit rotavirus replication in vitro and in vivo. These data provide that understanding the role of cellular miR-7 during rotaviral replication may help in the identification of novel therapeutic small RNA molecule drug for anti-rotavirus.