Phospholipase D alpha 1 (PLD alpha 1, AT3G15730) and mitogen-activated protein kinases (MAPKs) participate on signaling-dependent events in plants. MAPKs are able to phosphorylate a wide range of substrates putatively including PLDs. Here we have focused on functional regulations of PLD alpha 1 by interactions with MAPKs, their colocalization and impact on salt stress and abscisic acid (ABA) tolerance in Arabidopsis. Yeast two-hybrid and bimolecular fluorescent assays showed that PLD alpha 1 interacts with MPK3. Immunoblotting analyses likewise confirmed connection between both these enzymes. Subcellularly we co-localized PLD alpha 1 with MPK3 in the cortical cytoplasm close to the plasma membrane and in cytoplasmic strands. Moreover, genetic interaction studies revealed that pld alpha 1mpk3 double mutant was resistant to a higher salinity and showed a higher tolerance to ABA during germination in comparison to single mutants and wild type. Thus, this study revealed importance of new biochemical and genetic interactions between PLD alpha 1 and MPK3 for Arabidopsis stress (salt and ABA) response.