Abstract Study question To fill gaps in the evidence base on the autologous superior mitochondria donor cells, and its therapeutic effect on human embryo development among infertile population. Summary answer Autologous urine-derived mesenchymal stromal cells (USC) mitochondria transfer may be an effective strategy to improve embryonic development, especially in infertile females with advanced age. What is known already Mitochondria are vital for the initial development of oocytes and embryos. Transferring autologous mitochondria into oocytes of infertile females is a novel and feasible strategy for infertile problems without genomic safety and ethical concerns. Stem cells have biological advantages, but research and evidence in this area are quite scarce. Study design, size, duration In vitro fertilization/Intracytoplasmic sperm injection (IVF/ICSI) patients at the Assisted Reproductive Technology (ART) Centre of Peking University People’s Hospital (Beijing, China) were divided into young group (<35) and advanced age group (≥35) according to age. Oocytes obtained from each age group were randomly assigned to the conventional ICSI group and the mitochondria ICSI group. Participants/materials, setting, methods Mitochondrial morphology, mitochondrial DNA (mtDNA) copy number, mitochondrial activity, metabolic capacity are analyzed among autologous adipose, marrow, urine-derived mesenchymal stromal cells (ADSC, BMSC and USC) and ovarian germline granulosa cells (GC). Whole mitochondrial genome sequencing was performed to validate mtDNA biosecurity. The effect of mitochondrial transfer was evaluated by human early embryonic morphology, euploidy, mitochondrial content, mitochondrial membrane potential (MMP), cytosolic reactive oxygen species (ROS) and Ca2+ levels. Main results and the role of chance Among many types of human primary cells, urine-derived mesenchymal stromal cells (USC) demonstrate a non-fused spherical mitochondrial morphology and low oxidative stress status, which resembles the oocyte stage. Moreover, USC mitochondrial content, activity and function are all higher than other cell types and less affected by age, and it also exhibits a biphasic metabolic pattern similar to the pre-implantation stage of embryonic development. After the biosafety identification of USC mitochondrial genome, human early embryos after USC mitochondria transfer showed improvements in mitochondrial content, activity, and cytoplasmic Ca2+ levels. Further, aging embryos also showed improved embryonic morphological indicators, euploidy rates, and oxidative stress status. Limitations, reasons for caution Our study is an in-vitro basic research on human early embryos, further basic mechanisms and clinical trials are still needed to confirm its clinical therapeutic results. Wider implications of the findings Autologous USC mitochondria transfer provides evidence and possibility for the autologous treatment in the infertile females without invasive and ethical concerns. Trial registration number Beijing Natural Science Foundation (grant no. 7222197); State Key Program of the National Natural Science Foundation of China (grant no. 21737001)