Mammalian orthoreovirus (MRV) is an important zoonotic pathogen, but the regulatory role of its viral proteins in retinoic acid-inducible gene-like receptor (RLR)-mediated antiviral responses is still poorly understood. Herein, we show that MRV σ3 protein co-localizes with mitochondrial antiviral signaling protein (MAVS) in the mitochondria and promotes the mitochondria-mediated intrinsic apoptotic pathway to cleave and consequently degrade MAVS. Furthermore, tryptophan at position 133 of σ3 protein plays a key role in the degradation of MAVS. Importantly, we show that MRV outer capsid protein σ3 is a key factor in antagonizing RLR-mediated antiviral responses, providing evidence to better unravel the infection and transmission mechanisms of MRV.