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Although most osmotic stresses are harmful to plant growth and development, certain drought- or polyethylene glycol (PEG)-induced mild osmotic stresses promote plant root growth. The underlying regulatory mechanisms of this response remain elusive. Here, we report that the GLYCOGEN SYNTHASE KINASE 3 (GSK3) genes <i>ARABIDOPSIS THALIANA SHAGGY-RELATED KINASE 11</i> (<i>AtSK11</i>) <i>(AT5G26751</i>) and <i>AtSK12</i> (<i>AT3G05840</i>) are involved in the mild osmotic stress (−0.4 MPa) response in <i>Arabidopsis thaliana</i>. When grown on plant medium infused with different concentrations of PEG to mimic osmotic stress, both wild-type (WT) and <i>atsk11atsk12</i> plants showed stimulated root growth under mild osmotic stress (−0.4 MPa) but repressed root growth under relatively strong osmotic stress (−0.5, −0.6, −0.7 MPa) as compared to the mock condition (−0.25 MPa). The root growth stimulation of <i>atsk11atsk12</i> was more sensitive to −0.4 MPa treatment than was that of WT, indicating that <i>AtSK11</i> and <i>AtSK12</i> inhibit the mild stress-induced root growth response. RNA-seq analysis of WT and <i>atsk11atsk12</i> plants under three water potentials (−0.25 MPa, −0.4 MPa, −0.6 MPa) revealed 10 differentially expressed candidate genes mainly involved in cell wall homeostasis, which were regulated by <i>AtSK11</i> and <i>AtSK12</i> to regulate root growth in response to the mild stress condition (−0.4 MPa). Promoter motif and transcription factor binding analyses suggested that the basic helix-loop-helix (bHLH) transcription factor bHLH69/LJRHL1-LIKE 2 (LRL2) may directly regulate the expression of most −0.4 MPa-responsive genes. These findings indicate that mild osmotic stress (−0.4 MPa) promotes plant growth and that the GSK3 family kinase genes <i>AtSK11</i> and <i>AtSK12</i> play a negative role in the induction of root growth in response to mild osmotic stress.