A series of novel pyrano[3,2-a]phenazine derivatives (1a-1r and 2a-2q), designed as hybrid molecules of phenazine and pyran pharmocophores, were facilely synthesized in two steps with 77-93% overall yields in this study. Cytotoxic evaluation indicates that many compounds exhibited cytotoxicity against HCT116, MCF7, HepG2 and A549 cancer cell lines in vitro, in which compounds 1c, 1i, 2e, and 2l were found to have excellent antiproliferative activity against the HepG2 cancer cell line. Thus, inhibitory effect of subcutaneously implanted xenografted mice in vivo (H22H8D8 cells) of the four compounds as well as topoisomerase I and IIα inhibitory activities in vitro (HepG2 cells) were determined. Significantly, compound 1i showed more potent than positive control drug both in vivo and in vitro. Further mechanism studies against HepG2 cells in vitro revealed that compound 1i up-regulated the expression of both p53 and p21, which inhibited the expression of both cyclin B and CDK1, and arrested HepG2 cells in the G2/M phase. Concomitantly, after treating with compound 1i, Bax/Bcl-2 ratio was significantly increased, the cytochrome C was released from mitochondria to cytosol, and the cleavage of caspase-3 and caspase-9 expression levels were both increased. Together, all these evidences implicated that compound 1i acts as topoisomerase I and IIα dual inhibitor, cell cycle arrester and apoptosis inducer against HepG2 cells.