Objective To study whether human amniotic fluid colony derived stem cells (hAFCSCs) are involved in regeneration of injured muscles in mice and to investigate the method and feasibil ity of hAFCSCs-based cytotherapy in the treatment of injured muscles. Methods Human second-trimester amniotic fluid was collected through ultrasound-guided amniocentesis, hAFCSCs were isolated from second-trimester amniotic fluid and cultured, and the cells at 6th-8th passages were spared. The mRNA was extracted to identify the stem cell related genes by RT-PCR. The muscular injury model of bilateral tibial is anterior muscle was establ ished by cardiotoxin and X-ray irradiation in 16 Nod/Scid mice (aged 6-8 weeks, and weighing 20-24 g). The hAFCSCs (3.3 × 107/mL, 30 μL) were injected into the right injured tibial is anterior muscles as the experimental group, while the same volume of complete medium (α-MEM containing 15%FBS, 18%Chang B, 2%Chang C, 1% penicill instreptomycin, and 1% L-glutamine) was injected into the left injured tibial is anterior muscles as the control group. At 2 and 4 weeks after cell transplantation, the immunofluorescence staining of tibial is anterior muscles was performed to detect hepatocyte growth factor receptor (c-Met), myogenic regulatory factor (Myf-5), Laminin, Desmin, and human specific nuclear mitotic apparatus protein (NuMa). Results The clone formation was observed at 5-7 days of primary hAFCSCs culture; after 8-10 days, the clones with homogeneous morphology were selected for subculture. Adequate stem cells were available after 6th-8th subculture. RT-PCR analysis showed that hAFCSCs expressed mRNA of the stem cell related genes. The immunofluorescence double-staining showed that NuMa expressed in tibial is anterior muscles of the experimental group and no myogenic phenotype expressed at 2 weeks after cell transplantation, and that single cell co-expressed NuMa and c-Met or Myf-5 at 4 weeks after cell transplantation. In some myofibers, NuMa and Laminin or Desmin were also co-expressed. No NuMa positive hAFCSCs were detected in the control group at 2 and 4 weeks after cell transplantation. Conclusion hAFCSCs can participate in the regeneration of injured mouse muscle.