The aim of this study was to evaluate the advantages of the two-step embryo transfer (ET) strategy combining a day 2/3 ET with a day 5/6 blastocyst transfer. In an observational comparative study, 400 infertile women were enrolled from two assisted reproductive technology (ART) units according to inclusion criteria: age below 42 years and at least three embryos obtained on day 2 thus allowing an extended in vitro culture. Two groups were defined according to the ET strategy adopted: group 1 had a two-step ET; and group 2 had a day 2/3 ET with (subgroup 2a) or without (subgroup 2b) blastocysts cryopreserved on day 5/6. Live birth rate was significantly higher in group 1 than in subgroups 2a and 2b (36.5% versus 29.4% and 13.4%, respectively; p < 10−3). Multiple pregnancy rates were comparable between groups. After adjusting on major prognostic factors, the two-step ET strategy was still associated with a significantly higher live birth rate than the day 2/3 ET (OR = 2.23; 95% CI: 1.32–3.77). The two-step ET provides better live birth rates than the cleavage-stage ET. It does not increase multiple pregnancy rates if the number of embryos transferred is limited. It also prevents cycle loss when embryos fail to develop into blastocysts.