The volatile oil was extracted from water caltrop by steam distillation; it was then analyzed by GC-MS to obtain 16 components, 8 of which were identified. Apocynin was the most abundant one, accounting for 81.41% of the total oil. The in vitro inhibitory effects of the volatile oil on SMMC-7721, MCF-7, Hela, HL-60 cells, and human peripheral blood mononuclear celIs(PBMC) were investigated via the MTT method. The morphological changes of the tumor cells were observed and the apoptosis of HL-60 cells was detected by flow cytometry. The proliferation of the tumor cells could be significantly inhibited and the apoptosis of HL-60 cells could be induced by the volatile oil. The proliferation inhibition effect of the volatile oil on HL-60 tumor cells and the induction of the apoptosis of HL-60 cells had dose-dependent feature.