Abscisic acid (ABA) signaling plays important roles in plant growth, development and adaptation to various stresses. RCAR1/PYL9 has been known as a cytoplasm and nuclear ABA receptor in Arabidopsis. To obtain further insight into the regulatory mechanism of RCAR1/PYL9, a yeast two-hybrid approach was performed to screen for RCAR1/PYL9-interacting proteins and an R2R3-type MYB transcription factor, AtMYB44, was identified. The interaction between RCAR1/PYL9 and AtMYB44 was further confirmed by glutathione S-transferase (GST) pull-down and bimolecular fluorescence complementation (BiFC) assays. Gene expression analysis showed that AtMYB44 negatively regulated the expression of ABA-responsive gene RAB18, in contrast to the opposite role reported for RCAR1/PYL9. Competitive GST pull-down assay and analysis of phosphatase activity demonstrated that AtMYB44 and ABI1 competed for binding to RCAR1/PYL9 and thereby reduced the inhibitory effect of RCAR1/PYL9 on ABI1 phosphatase activity in the presence of ABA in vitro. Furthermore, transient activation assay in protoplasts revealed AtMYB44 probably also decreased RCAR1/PYL9-mediated inhibition of ABI1 activity in vivo. Taken together, our work provides a reasonable molecular mechanism of AtMYB44 in ABA signaling.