Objective: To determine whether oocyte activation using a combination of calcium ionophore A23187 (A23187) with puromycin could salvage human unfertilized oocytes after ICSI. Study Design: One hundred and thirteen discarded unfertilized oocytes 20–68 h after ICSI were assigned to four roups: ICSI 20-h group, ICSI 44-h group, ICSI 68-h group and control. All unfertilized oocytes were exposed to A23187 (5 mM) for 5 min and subsequently were incubated with puromycin (10 mg/ml) for 4 h. Sex chromosomal analysis was performed by dual color fluorescence in situ hybridization (FISH). Results: The combination of A23187 with puromycin could activate the unfertilized oocytes 20–68 h after ICSI. The best results were achieved in the ICSI 20-h group, which exhibited an activation rate of 91.2% (31/34), a cleavage rate of 64.7% (22/34) and 44.1% (15/34) high-quality embryos. The activation rate, cleavage rate and the number of high-quality embryos appeared to decrease with the cultured time of unfertilized oocytes after ICSI. FISH analysis showed six embryos with XX and seven embryos with XY in 16 embryos derived from 2PN2PB. Conclusions: The combination of calcium ionophore A23187 with puromycin could effectively salvage unfertilized oocytes within 20 h after ICSI.