Picornavirus positive-strand RNAs are selectively encapsidated despite the coexistence of viral negative-strand RNAs and cellular RNAs in infected cells. However, the precise mechanism of the RNA encapsidation process in picornaviruses remains unclear. Here we report the first identification of an RNA element critical for encapsidation in picornaviruses. The 5' end of the genome of Aichi virus, a member of the family Picornaviridae, folds into three stem-loop structures (SL-A, SL-B, and SL-C, from the most 5' end). In the previous study, we constructed a mutant, termed mut6, by exchanging the seven-nucleotide stretches of the middle part of the stem in SL-A with each other to maintain the base pairings of the stem. mut6 exhibited efficient RNA replication and translation but formed no plaques. The present study showed that in cells transfected with mut6 RNA, empty capsids were accumulated, but few virions containing RNA were formed. This means that mut6 has a severe defect in RNA encapsidation. Site-directed mutational analysis indicated that as the mutated region was narrowed, the encapsidation was improved. As a result, the mutation of the 7 bp of the middle part of the stem in SL-A was required for abolishing the plaque-forming ability. Thus, the 5'-end sequence of the Aichi virus genome was shown to play an important role in encapsidation.