Although, it is well known that pre-incubation of oocytes prior to conventional IVF improves fertilization and pregnancy rates, there are conflicting results regarding the effect of pre-incubation time in ICSI. This study evaluated the role of pre-incubation of oocytes on outcome in intracytoplasmic sperm injection (ICSI) cycles. A total of 1260 patients undergoing their first ICSI cycles were evaluated retrospectively. In patients undergoing ICSI during the year 2000 (Group I, n = 670), oocytes were injected immediately after retrieval, whereas in patients undergoing ICSI during 2001 (Group II, n = 590), oocytes were incubated for 2–4 h prior to injection. The mean age of patients was 33.9 ± 5.04 years and 34.1 ± 5.06 years in groups I and II, respectively. The number of oocytes with a first polar body (MII) and fertilization and cleavage rates were higher, and embryo quality was significantly better in group II. In contrast, the total numbers of oocytes without a first polar body (MI), those where germinal vesicle breakdown had not occurred (GV), and empty zona oocytes were higher in group I. No difference was found in the number of embryos transferred or implantation or clinical pregnancy rates. This study demonstrated that pre-incubation of oocytes prior to ICSI is associated with improved maturation of oocytes, fertilization and embryo quality.