Arbuscular mycorrhizal (AM) fungi are important in plant resistance against insects and disease. However, the effect of AM fungi in plants during simultaneous pathogen and insect attacks is unknown. Phoma medicaginis and pea aphid (Acyrthosiphon pisum) majorly limit alfalfa production worldwide. This study established an alfalfa (Medicago sativa)-AM fungus Rhizophagus intraradices-P. medicaginis-pea aphid system. RNA-seq analysis was done to determine the differentially expressed genes, plant defense, and signaling substances in different alfalfa treatments.Pea aphid increased P. medicaginis disease occurrence by 24.94%. The AM fungus decreased the disease index by 22.37% and enhanced alfalfa growth and development by increasing the plant total N and total P uptake. P. medicagonis induced plant CAT and POD activity by 292.93 and 63.46%, NO and trypsin inhibitor contents by 9.80 and 5.71%. However, aphids induced PPO activity, and AM fungi enhanced plant-defense enzyme activity against aphid infestation and subsequent P. medicaginis infection. Additionally, the AM fungus increased the SA and ABA content of plants exposed to pathogen- or aphid-stress. Abscisic acid and hormone binding GO terms were up-regulated in aphid-infested or pathogen-infected alfalfa. However, AM fungus down-regulated genes related to the KEGG pathway, ribosome, for resisting P. medicaginis and pea aphid attack. The MAPK signaling pathway-plant (map04016) was up-regulated in alfalfa against pea aphid or P. medicaginis.Moreover, the AM fungus enhanced aphid-induced plant defense and signaling and may defend alfalfa against subsequent disease and insect pest attacks.