Seven novel 4-amino acid derivative substituted pyrimidine nucleoside analogues were designed, synthesized, and tested for their anti-CVB3 activity. Initial biological studies indicated that among these 4-amino acid derivative substituted pyrimidine nucleoside analogues, 4-N-(2'-amino-glutaric acid-1'-methylester)-1-(2'- deoxy-2'-β-fluoro-4'-azido)-furanosyl-cytosine 2 exhibited the most potent anti-CVB activity (IC<sub>50</sub> = 9.3 μM). The cytotoxicity of these compounds has also been assessed. The toxicity of compound 2 was similar to that of ribavirin.