Enterovirus 71 (EV-71) is a major pathogenic factor that causes hand, foot, and mouth disease in young children and infants. Given the limited treatments for EV-71 infection, discovering new host factors and understanding the mechanisms involved will aid in combating this viral infection. Neutral sphingomyelinase-2 (nSMase-2, encoded by SMPD3) is a crucial cellular cofactor in viral infection. We found that EV-71 infection increased nSMase-2 expression in African green monkey kidney cells (Vero cells). Knockdown of nSMase-2 by small interfering RNA obviously decreased the viral replication and infectivity. Furthermore, the knockdown of nSMase-2 reduced autophagy-associated proteins expression. Collectively, our findings uncovered a potential mechanism of nSMase-2 supporting EV-71 infection.