Depression is a prevalent neuropsychiatric disorder characterized by persistent sadness and a lack of interest, significantly impacting the quality of life. Icaritin, a bioactive compound from Herba Epimedii, which has the antidepressant-like effects. However, the potential target of icaritin in the brain, especially concerning transcription factors, is not well understood. In this study, we demonstrated that icaritin significantly ameliorated depressive-like behaviors in a chronic corticosterone (CORT)-induced mouse model. This study aimed to investigate the role of icaritin in modulating the activity of cAMP-response element binding protein 1 (CREB1), a crucial transcription factor implicated in neuronal function and mood regulation. Through a combination of virtual screening and molecular docking, we identified CREB1 as a target by intersections of databases, leading to the selection of 43 candidate compounds, among which icaritin exhibited a favorable docking score and previously reported antidepressant effects. Biolayer interferometry (BLI) analysis confirmed the direct binding of icaritin to CREB1, with a dissociation constant (K