Stress granules (SGs) contain stalled messenger ribonucleoprotein complexes, and are related to the regulation of mRNA translation. Picornavirus infection can interfere with the formation of SGs. However, the molecular mechanisms of SG assembly and picornavirus-mediated inhibition of SG formation are not clear. Here, we show that G3BP-eIF4GI interaction is critical for typical SG formation, and protease 2A of EV71 disrupted G3BP-eIF4GI interaction by cleaving eIF4GI and inhibiting the RNA binding of G3BP to block typical SG formation. Meanwhile, 2A also induced the formation of aberrant SGs by cleavage of eIF4GI, which selectively confined those energy-exhausting cellular mRNAs. Moreover, we show that typical SGs formed in cells infected with EV71 recombinant containing the 2AC110S mutation and sequestered viral and cellular mRNAs. Thus, we propose a model revealing 2A-mediated disruption of typical SG formation and induction of aberrant SG formation in the process of picornavirus infection to facilitate viral translation.