Choline (Cho) is an essential nutrient for humans as well as the precursor of glycine betaine (GlyBet), an important compatible solute in eukaryotes that protects cells from osmotic stress caused by dehydrating conditions. The key enzyme for plant Cho synthesis is phosphoethanolamine N-methyltransferase (PEAMT), which catalyzes all three methylation steps, including the rate-limiting Nmethylation of phosphoethanolamine. Herein, we report that the beneficial soil bacterium Bacillus subtilis (strain GB03) enhances Arabidopsis Cho and GlyBet synthesis associated with enhanced plant tolerance to osmotic stress. When stressed with 100 mM exogenous mannitol, GB03-exposed plants exhibit increased transcript level of PEAMT compared with stressed plants without bacterial exposure. Endogenous Cho and GlyBet metabolite pools were elevated by more than two- and fivefold, respectively, by GB03 treatment, consistent with increased stress tolerance. Moreover, in the xipotl mutant line with reduced Cho production, a loss of GB03-induced drought tolerance is observed. Osmotic-stressed plants with or without GB03 exposure show similar levels of abscsisic acid (ABA) accumulation in both shoots and roots, suggesting that GB03-induced osmoprotection is ABA independent. GB03 treatment also improves drought tolerance in soil-grown plants as characterized by phenotypic comparisons, supported by an elevated accumulation of osmoprotectants. These results provide a biological strategy to enhance Cho biosynthesis in plants and, in turn, increase plant tolerance to osmotic stress by elevating osmoprotectant accumulation.