Background <p>The emergence of drug resistance poses a formidable obstacle in the treatment of breast cancer (BC). Doublecortin-like kinase 1 (DCLK1) has been identified as a tumor promoter in BC. However, the impact of DCLK1 on adriamycin (ADM) resistance in BC remains largely unknown.</p> Methods <p>The factors linked to ADM resistance in BC, the interaction between DCLK1 and STIP1 homology and U-box containing protein 1 (STUB1), and the binding sequence between Sp1 transcription factor (SP1) and DCLK1 were predicted by bioinformatics. The impact on cell phenotypes was evaluated by measuring the IC₅₀ value for ADM, cell apoptosis, proliferation, viability, invasion, and migration. The impact on tumor growth was tested using subcutaneous xenograft studies. The STUB1/DCLK1 relationship was validated by IP assay and protein stability analysis. The SP1/DCLK1 relationship was confirmed by chromatin immunoprecipitation (ChIP) and luciferase assays.</p> Results <p>DCLK1 was upregulated in ADM-resistant BC tissues and cell lines. DCLK1 depletion impaired cell proliferation, invasion, and migration and sensitized them to ADM in vitro, as well as enhanced the sensitivity of subcutaneous xenografts to ADM. Mechanistically, STUB1 degraded DCLK1 protein through ubiquitination, and SP1 transcriptionally increased DCLK1 expression. DCLK1 upregulation reversed the effects of STUB1 on ADM sensitivity and malignant behaviors of ADM-resistant BC cells, and DCLK1 expression restoration reversed the impact of SP1 silencing.</p> Conclusion <p>Our findings have identified DCLK1 as a crucial regulator in the malignant phenotypes and ADM sensitivity of ADM-resistant BC cells, providing a potential target for enhancing the anti-cancer efficacy of ADM in BC.</p>