<p>Background: Although rescue intracytoplasmic sperm injection (r-ICSI) is extensively used worldwide, the indication of r-ICSI and its optimal timing remains obscure. This study aimed to assess the outcomes of r-ICSI following in vitro fertilization in different timings when fertilization is confirmed.<br /> Methods: This study included 5,156 cycles (47,785 eggs). Fertilization was confirmed by polar body analysis after 4 and 6 <em>hr</em> of coincubation of the sperm and oocyte. Oocytes that underwent IVF were divided into two groups based on the time when a second polar body was detected in more than 30% of all oocytes (Four-<em>hr</em> group and six-<em>hr</em> group). If the second polar body was not detected or was present in less than 30% of all oocytes after six <em>hr</em> of coincubation, rescue-ICSI (r-ICSI) was performed for oocytes without a second polar body (r-ICSI group).<br /> Results: The fertilization rates of two pronuclear (2PN) oocytes in the three groups (Four-<em>hr</em> group, six-<em>hr</em> group, and r-ICSI group) were 70.7%, 51.3%, and 58.0%, respectively. The blastocyst formation rates were 62.8%, 53.4%, and 42.9%, respectively.<br /> Conclusion: Performing r-ICSI after six <em>hr</em> of coincubation can salvage cases with fertilization failure in IVF. The higher fertilization rate of r-ICSI indicates that all oocytes without signs of fertilization after six <em>hr</em> of coincubation should undergo r-ICSI.</p>