The homeodomain-leucine zipper (HD-Zip) proteins play crucial roles in plant developmental and environmental responses. However, how they mediate gene expression to facilitate abiotic stress tolerance re-mains unknown. In the present study, we characterized BpHOX2 (encoding a HD-Zip I family protein) from birch (Betula platyphylla). BpHOX2 is predominately expressed in mature stems and leaves, but expressed at a low level in apical buds and roots, suggesting that it has tissue-specific characteristics. BpHOX2 expression was highly induced by osmotic and salt, but only slightly induced by abscisic acid. Overexpression of BpHOX2 markedly improved osmotic tolerance, while knockdown of BpHOX2 increased sensi-tivity to osmotic stress. BpHOX2 could induce the ex-pression of pyrroline-5-carboxylate synthase, peroxidase, and superoxide dismutase genes to improve proline levelsand the reactive oxygen species scavenging capability. Chromatin immunoprecipitation sequencing combined with RNA sequencing showed that BpHOX2 could bind to at least four cis-acting elements, including dehydration-responsive element“RCCGAC”, Myb-p binding box“CCWACC,”and two novel cis-acting elements with the sequences of“AAGAAG”and“TACGTG”(termed HBS1 and HBS2, respectively) to regulate gene expression. Our results suggested that BpHOX2 is a transcription factor that binds to different cis-acting elements to regulate gene expression, ultimately improving osmotic tolerance in birch.