A series of novel N-1-monoallylated 2,5-diketopiperazine derivatives were designed, synthesized, and evaluated as cytotoxic agents against eight cancer cell lines by using CCK8 assay. These derivatives were substituted with methoxyphenyl groups at C-6 position, and various long alkyl side chains at C-3-position of the 2,5-diketopiperazine ring. The cytotoxic results showed that 4-methoxyphenyl group was better than 2-methoxyphenyl group as optimal substitutive group, while 3-methoxyphenyl group was not a suitable one. When the number (n value) of the methylene groups for the long alkyl side chain was 3 (compounds 1c and 3c), the derivatives had the strongest cytotoxicities. Compound 3c substituted with 4-methoxyphenyl group and pentylidene side chain exhibited strong activity (IC50 = 0.36-1.9 μM) against all cancer cell lines, and could obviously induce apoptosis of cancer cell line U937 at 1.0 μM after 48 h treatment.