Methyl 2-(5-fluorouracil-1-yl)acetamidoactate was synthesized at room temperature by liquid coupling method with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 1-Hydroxybenzotriazole (HOBt) as a condensation reagent, 5-fluorouracil as a starting material, in which the yield was 72%. 2-(5-fluorouracil-1-yl)acetamidoactic acid was obtained by hydrolysis under pH 10-11 and 20-30 ℃ with the yield 75%. The structures of the two compounds were confirmed by 1H NMR, 13C NMR, mass spectroscopy and infrared spectroscopy etc. The antitumor mechanism of the compounds was initialy revealed by UV spectra. The compounds’ in vitro antitumor activities against HL-60 and BEL-7402 were tested by The National Center for Drug Screening. The results indicated that the compounds have some antitumor effect in vitro.