Abscisic acid (ABA) is an important phytohormone mediating osmotic stress responses. SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASE 2.6 (SnRK2.6, also named OPEN STOMATA1 and SNF1-RELATED KINASE 2E) is central in the ABA signaling pathway; therefore, manipulating its activity may be useful to confer stress tolerance in plants. Pladienolide B (PB) is an mRNA splicing inhibitor and enhances ABA responses. Here, we analyzed the effect of PB on Arabidopsis SnRK2.6. PB enhanced the activity of recombinant SnRK2.6 in vitro through direct physical interaction as predicted by molecular docking simulations followed by mutation experiments and isothermal titration calorimetry. Structural modeling predicted probable interaction sites between PB and SnRK2.6, and experiments with mutated SnRK2.6 revealed that Leu-46 was the most essential amino acid residue for SnRK2.6 activation by PB. This study demonstrates the feasibility of SnRK2.6 chemical manipulation and paves the way for the modification of plant osmotic stress responses.