Reversible protein phosphorylation mediated by protein kinases and phosphatases plays important roles in the regulation of leaf senescence. We previously reported that the senescence-associated leucine-rich repeat receptor-like kinase AtSARK autophosphorylates on both serine/threonine and tyrosine residues and functions as a positive regulator of Arabidopsis leaf senescence; the senescence-suppressed protein phosphatase SSPP interacts with and dephosphorylates the cytoplasmic domain of AtSARK, thereby negatively regulating leaf senescence. Here, 27 autophosphorylation residues of AtSARK were revealed by mass spectrometry analysis, and six of them, including two Ser, two Thr, and two Tyr residues, were further found to be important for the biological functions of AtSARK. All site-directed mutations of these six residues that resulted in decreased autophosphorylation level of AtSARK could significantly inhibit AtSARK-induced leaf senescence. In addition, mutations mimicking the dephosphorylation form of Ser384 (S384A) or the phosphorylation form of Tyr413 (Y413E) substantially reduced the interaction between AtSARK and SSPP. All results suggest that autophosphorylation of AtSARK is essential for its functions in promoting leaf senescence. The possible roles of S384 and Y413 residues in fine-tuning the interaction between AtSARK and SSPP are discussed herein.