A series of novel pyrazolo[1,5-a]pyrimidine derivatives bearing nitrogen mustard moiety were designed, synthesized and evaluated for their antiproliferative activities against five human cancer cell lines (A549, SH-SY5Y, HepG2, MCF-7 and DU145) in vitro. Among these compounds, 13b exhibited potent inhibitory effect on the proliferation of the five tumor cells and was able to inhibit cell cycle arrest at G1 phase and induce cell apoptosis. In HepG2 HCC xenograft compound 13b was selected for evaluating the antitumor activity in vivo which exhibited significant cancer growth inhibition with low host toxicity in vivo.