InArabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by a family of six genes (ACBP1toACBP6), and are essential for diverse cellular activities. Recent investigations suggest that the membrane-anchored ACBPs are involved in oxygen sensing by sequestration of group VII ethylene-responsive factors under normoxia. Here, we demonstrate the involvement of Arabidopsis ACBP3 in hypoxic tolerance.ACBP3transcription was remarkably induced following submergence under both dark (DS) and light (LS) conditions.ACBP3-overexpressors (ACBP3-OEs) showed hypersensitivity to DS, LS and ethanolic stresses, with reduced transcription of hypoxia-responsive genes as well as accumulation of hydrogen peroxide in the rosettes. In contrast, suppression ofACBP3inACBP3-KOsenhanced plant tolerance to DS, LS and ethanol treatments. By analyses of double combinations ofOE-1withnpr1-5,coi1-2,ein3-1as well asctr1-1mutants, we observed that the attenuated hypoxic tolerance inACBP3-OEswas dependent on NPR1- and CTR1-mediated signaling pathways. Lipid profiling revealed that both the total amounts and very-long-chain species of phosphatidylserine (C42:2- and C42:3-PS) and glucosylinositolphosphorylceramides (C22:0-, C22:1-, C24:0-, C24:1-, and C26:1-GIPC) were significantly lower inACBP3-OEsbut increased inACBP3-KOsupon LS exposure. By microscale thermophoresis analysis, the recombinant ACBP3 protein bound VLC acyl-CoA esters with high affinitiesin vitro. Further, a knockout mutant of MYB30, a master regulator of very-long-chain fatty acid (VLCFA) biosynthesis, exhibited enhanced sensitivities to LS and ethanolic stresses, phenotypes that were ameliorated byACBP3-RNAi. Taken together, these findings suggest that Arabidopsis ACBP3 participates in plant response to hypoxia by modulating VLCFA metabolism.