A series of new hydroxylated chalcone derivatives with different substitution patterns on a Ph ring A and B, were prepared by Claisen-Schmidt condensation in an aqueous alk. base. The antiproliferative activity of the studied compounds was evaluated against the human leukemia cell line U-937. The structure-activity relationship of these naphthylchalcones was investigated by the introduction of one methoxy or two Me groups on the A ring, the introduction of a methoxy group on the naphthyl ring or by varying the position of the methoxy group on the A ring. The results revealed that the naphthylchalcone containing a methoxy group in position 6 of the A ring was the most cytotoxic compound, with an IC50 value of 4.7 ± 0.5μM against U-937 cells. This synthetic chalcone induced S and G2-M cell cycle arrest, a time-dependent increase in sub-G1 ratio and annexin-V pos. cells, caspase activation and poly(ADP-ribose) polymerase cleavage. Apoptosis induction was blocked by a pan-caspase inhibitor and by the selective caspase-3/7 inhibitor and attenuated by the inhibition of c-jun N-terminal kinases / stress-activated protein kinases (JNK/SAPK) and phosphoinositide 3-kinase. The structure-activity relationship of naphthylchalcones against human leukemia cells reveals that the major determining in cytotoxicity is the presence of a methoxy group in position 6 of the A ring that suggest the potential of this compound or derivatives in the development of new anti-leukemia drugs.