Acetoin is a volatile organic compound, which is a class of metabolites produced by plant growth-promoting rhizobacteria. The mechanisms underlying plant growth promotion by acetoin and its potential to induce saline stress tolerance in plants are poorly understood. Lettuce (Lactuca sativa L. var. ramosa Hort.) seedlings in hydronics and pots under non-saline or saline conditions were foliar-sprayed with 10 mL of 0 or 1 mg·mL−1 acetoin at 7 and 14 d after transplantation and harvested 7 d after the second spray. Shoots and roots of hydroponic lettuce seedlings were harvested at 6 and 24 h after treatment for RNA sequencing. Seedlings sprayed with acetoin showed more vigorous growth, with higher shoot and root biomass than those of the controls, in both hydronic and pot modes. The transcriptomic analysis revealed acetoin application resulted in 177 differentially expressed genes (39 upregulated and 138 downregulated) in shoots and 397 differentially expressed genes (112 upregulated and 285 downregulated) in roots. These DEGs, mainly involved in plant hormone signal transduction and the mitogen-activated protein kinase, have the potential to trigger plants’ responses to various environmental stimuli, including stress and developmental signals. Under saline conditions, acetoin-treated plants showed increased net leaf photosynthesis and activities of several defense enzymes, indicating that acetoin enhances both fundamental growth and the plant’s stress defenses, especially against salinity. In summary, acetoin appears to act through a complex interplay of genetic and biochemical mechanisms, influencing key signaling pathways and physiological processes that lead to improved growth and stress tolerance in lettuce seedlings.