Trihelix transcription factors are characterized by containing a conserved trihelix (helix-loop-helix-loop-helix) domain that bind to GT elements required for light response, play roles in light stress, and also in abiotic stress responses. However, only few of them have been functionally characterised. In the present study, we characterized the function of AST1 (Arabidopsis SIP1 clade Trihelix1) in response to abiotic stress. AST1 shows transcriptional activation activity, and its expression is induced by osmotic and salt stress. The genes regulated by AST1 were identified using qRT-PCR and transcriptome assays. A conserved sequence highly present in the promoters of genes regulated by AST1 was identified, which is bound by AST1, and termed AGAG-box with the sequence [A/G][G/A][A/T]GAGAG. Additionally, AST1 also binds to some GT motifs including GGTAATT, TACAGT, GGTAAAT and GGTAAA, but failed in binding to GTTAC and GGTTAA. Chromatin immunoprecipitation combined with qRT-PCR analysis suggested that AST1 binds to AGAG-box and/or some GT motifs to regulate the expression of stress tolerance genes, resulting in reduced reactive oxygen species, Na+ accumulation, stomatal apertures, lipid peroxidation, cell death and water loss rate, and increased proline content and reactive oxygen species scavenging capability. These physiological changes mediated by AST1 finally improve abiotic stress tolerance.