The present study evaluated the effect of artificial oocyte activation (AOA) with calcium ionophore A23187 on intracytoplasmic sperm injection (ICSI) cycles using spermatozoa from different sources. The 314 cycles evaluated were divided into three groups according to sperm origin; the ejaculated group (n = 92), the epididymal group (n = 82), and the testicular group (n = 140). Each group was further split into experimental subgroups, depending on whether or not AOA was performed. In addition, the cycles of women younger than 36 years were evaluated separately. For each experimental group, ICSI outcomes were compared between subgroups. No significant difference was observed between subgroups for all sperm origin groups. When evaluating only the cycles of women younger than 36 years of age, AOA increased the percentage of high-quality embryos (74.5 versus 53.0%, P = 0.011) and the implantation rate (19.3 versus 10.5%, P = 0.0025) when it was used with ejaculated spermatozoa, and the percentage of high-quality embryos (64.4 versus 50.3%, P = 0.006) when epididymal spermatozoa were used. These results may suggest that both sperm maturity and oocyte quality play a role in oocyte activation. However, this study is to be continued to confirm these findings.