Purpose <p>To investigate whether the one-step warming (OW) procedure affects oocyte survival and developmental potential after fertilization in vitrified oocytes.</p> Methods <p>Standard vitrified mouse oocytes were warmed using two different procedures: (1) standard multi-step warming (SW) and (2) one-step warming (OW). The survival rates and early embryonic development potentials of the warmed oocytes were compared following insemination by piezo-ICSI. Spindle integrity of the warmed oocytes was evaluated by α-tubulin/Hoechst staining, and mitochondrial status was assessed using Mitotracker/JC-1 staining, with mitochondrial damage analyzed by Confocal microscope. Additionally, the apoptosis status of the resulting blastocysts in each group was compared using a TUNEL assay.</p> Results <p>The OW procedure maintained oocyte survival and fertilization rates comparable to those of the SW procedure, but significantly (<i>P</i> &lt; 0.05) increased blastocyst development after fertilization. Mechanistic measures revealed that the OW procedure improved embryonic developmental potential by mitigating mitochondrial damage compared with the SW procedure.</p> Conclusions <p>These findings provide a theoretical foundation for optimizing the OW procedure for already vitrified and stored oocytes in ARTs.</p>