Two series of more available novel 1,2,3-triazole-Jaspine B hybrids were efficiently synthesized employing click chemistry approach and evaluated for their cytotoxic activities against three human cancer cell lines (EC-9706, MGC-803 and MCF-7). Among them, compound 14h showed excellent inhibition against MCF-7 (IC50 = 1.93 μM) and was more potent than 5-Fu and Jaspine B against all three cancer cell lines. Further investigation of apoptosis assay and cell cycle analysis demonstrated that compound 14h caused cellular early and late apoptosis and arrested the cell cycle at G2/M phase in a concentration- and time-independent manner. This was the first report about the synthesis and in vitro cytotoxic evaluation of 1,2,3-triazole-Jaspine B hybrids.