AbstractPepper mild mottle virus (PMMoV) is a devastating viral pathogen of pepper (Capsicum annuum) but it is unclear whether and how peppers protect against PMMoV infection. The expression of the chloroplast outer membrane protein 24 (OMP24) ofC. annuumwas upregulated under PMMoV infection and it interacted with PMMoV coat protein (CP). Silencing ofOMP24in eitherC. annuumorNicotiana benthamianafacilitated PMMoV infection, whereas overexpression ofN. benthamiana OMP24in transgenic plants inhibited PMMoV infection. BothC. annuumOMP24 (CaOMP24) andN. benthamianaOMP24 (NbOMP24) localized to the chloroplast and have a moderately hydrophobic transmembrane domain that is necessary for their localization. Overexpression ofCaOMP24induced stromules, perinuclear chloroplast clustering, and accumulation of reactive oxygen species (ROS), the typical defense responses of chloroplasts transferring the retrograde signaling to the nucleus to regulate resistance genes. The expression ofPR1andPR2was also upregulated significantly in plants overexpressingOMP24. Self-interaction of OMP24 was demonstrated and was required for OMP24-mediated plant defense. Interaction with PMMoV CP interfered with the self-interaction of OMP24 and impaired OMP24-induced stromules, perinuclear chloroplast clustering and ROS accumulation. The results demonstrate the defense function of OMP24 in pepper during viral infection and suggest a possible mechanism by which PMMoV CP modulates the plant defense to facilitate viral infection.