Six previously undescribed coumarins (1-6), including one pair of enantiomers, along with 23 known coumarin derivatives (7-29), were isolated from the branches and leaves of Zanthoxylum dimorphophyllum var. spinifolium. Their structures were elucidated through comprehensive spectroscopic analysis, including HR-ESIMS, NMR, and ECD. These compounds were subsequently evaluated for their antileukemic activity. Among them, the previously undescribed compound 5 exhibited significant antiproliferative activity against three cancer cell lines: erythroleukemia (HEL), promyelocytic (HL-60), and chronic myelogenous leukemia (K562), with IC50 values of 30.5 ± 1.2, 10.2 ± 0.7, and 19.8 ± 1.2 μM, respectively. HL-60 cells were the most sensitive to compound 5, exhibiting the lowest IC50 value of 10.2 ± 0.7 μM. Flow cytometric analysis revealed that compound 5 induced apoptosis in HL-60 cells in a dose-dependent manner and induced significant cell cycle arrest at the S phase. Furthermore, in a zebrafish xenograft model, compound 5 significantly inhibited the proliferation of HL-60 cells in vivo.