At our clinic, a successfully fertilized human oocyte is defined as one with two polar bodies and two pronuclei (PN) 18 h after ICSI, but when some oocytes do not form PN. Recently, a successful pregnancy and live birth following artificial oocyte activation (AOA; using Ca² ionophore) of ICSI-treated one-day-old unfertilized oocytes was reported. In this study, we performed AOA on PN-lacking unfertilized oocytes, 18 h after ICSI (defined as Day 1-AOA, since ICSI=Day 0). The abilities of these oocytes to form PN and develop were compared with those of oocytes fertilized by ICSI (defined as No-AOA). Piezo-ICSI was performed on 168 oocytes, 50 of which failed to fertilize (0PN) but were subsequently artificially activated. 2PN formation rate was significantly lower in Day 1-AOA, and the rates of 1PN and ≥4PN were significantly higher than in No-AOA. In Day 1-AOA, 8 oocytes formed 2PN, 6 zygotes underwent cleavage and 2 of them developed to the morula stage, but none formed blastocysts. The rates of cleavage, morula, and blastocyst formation were significantly lower in Day 1-AOA than in No-AOA. These results suggest that the AOA protocol has room for improvement in the activation of unfertilized oocytes 18 h after ICSI.