Purpose <p>To investigate whether single frozen-thawed blastocyst transfers derived from poor-grade Day 3 embryos influence neonatal outcomes.</p> Methods <p>This retrospective cohort study conducted in a tertiary academic medical center included all the live-born singletons (<i>n</i> = 3156) in vitrified-warmed single blastocyst transfer cycles from January 2013 to November 2023, which were divided into the poor-grade group (<i>n</i> = 2432) and the high-grade group (<i>n</i> = 724) based on the embryo grade on Day 3. Propensity score matching (PSM) analysis and multivariate regression analysis were used to explore the association between the embryo grade on Day 3 and neonatal outcomes.</p> Results <p>After PSM, the baseline characteristics were all comparable between the two groups. There was a significant gender difference between the two groups (<i>P</i> = 0.004), while there were no differences in the mode of delivery, very preterm birth, preterm birth, very low birth weight, low birth weight, macrosomia, small for gestational age, large for gestational age, or congenital malformations (<i>P</i> &gt; 0.05). After multivariate regression analysis, it was shown that the proportion of male neonates born through blastocyst transfer derived from poor-grade Day 3 embryos was considerably higher (aOR 0.710, 95%CI 0.570–0.890; <i>P</i> = 0.003), whereas no differences were found in other outcome variables (<i>P</i> &gt; 0.05).</p> Conclusion <p>In the single frozen-thawed blastocyst transfer cycle, the poor-grade embryo on Day 3 is associated with a higher proportion of male neonates, but not with adverse neonatal outcomes.</p>