Microorganisms produce volatile organic compounds (VOCs) that have biological impacts on plants
however, it is unknown how these molecules participate in plants' responses to abiotic stress. This study aimed to determine the potential benefit of 3-methyl-1-butanol (3 MB), a microbial VOC, in helping rice (Oryza sativa) seedlings suffering from salinity stress. Our study revealed that rice seedlings primed with microbial volatile 3 MB for 12 h before exposure to salinity stress could decrease reactive oxygen species (ROS) generation and cell damage in rice roots. Additionally, antioxidant systems such as peroxidase (POD) isozymes 4 and 5 and catalase 1 (CAT1) increased after treatment with 3 MB + NaCl. The microbial volatile 3 MB fumigation also raised the proline content and activated the proline-related genes under 3 MB + NaCl treatment. To further elucidate the molecular mechanisms by which 3 MB assists rice in tolerating salinity stress, transcriptomic analysis was used to investigate the genome-wide gene expressions. Totally, 287 up-regulated differentially expressed genes (DEGs) were found. They are associated with phytohormone regulation, transcription factors, redox signaling, and defense responses. Through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and MapMan enrichment results of DEGs revealed that 3 MB could activate antioxidant systems, jasmonic acid (JA) pathway, and starch biosynthesis to generate more ATP, thus building a line of defense in response to salinity stress. This study provides valuation information indicating that microbial volatile 3 MB vapor can enhance salt stress tolerance in rice seedlings and clarify its underlying mechanism.