The functions of plant protein kinase SnRK1s are regulated by many factors. In this work, we used protein kinase GsSnRK1.1 as bait to screen the wild soybean cDNA libray and identified GsPP2CA and GsPKA as interactors of GsSnRK1.1 by yeast twohybrid. To further investigate the mechanism of how GsPP2CA and GsPKA regulate GsSnRK1.1 kinase activity, we expressed and purified these recombinant proteins in E.coli system for pulldown and phosphorylation assays. The results of pulldown analyses indicated that GsSnRK1.1 had physical interactions with GsPP2CA and GsPKA, and the Phostag assays and western blottings using specific pPKDsub antibody showed that the Thr176 of GsSnRK1.1 could be phosphorylated by GsPKA and could be dephosphorylated GsPP2CA. When we complemented these genes into ARY330 (snf1/reg1/sit4) yeast mutant strain, we found that GsSnRK1.1 could play the function of SNF1 kinase to make yeast cells grow on the media with nonfermented sugars. The phosphorylation level of GsSnRK1.1 protein kinase can be regulated by GsPP2CA and GsPKA, which may replace Reg1 and Sit4 in yeast and further to regulate the use of nonfermentative carbon sources by GsSnRK1.1.