Background: Recently, because of the demand for cell therapy, stem cells researches have been increased. Improvement in stem cell proliferation promotes the use of these cells in cellular treatments. Objectives: The aim of this study was to improve the culture conditions of adipose-derived stem cells (ADSCs) by treatment of these cells with a nontoxic concentration of ciprofloxacin. Methods: ADSCs were prepared from the human and animal cell bank of Iranian Biological Resource Center. The microbial contamination of cells including bacteria, fungi, and yeast had been tested. Adipogenic and osteogenic differentiation potential of ADSCs was demonstrated and the expression of surface markers including CD45, CD29, CD90, CD34, and CD105 was confirmed by flow cytometry. In the presence of ciprofloxacin at different concentrations, the viability of the cells was measured by MTT assay and the expression of the Oct-4, Nanog and GAPDH were examined by real-time PCR in a nontoxic concentration of ciprofloxacin-treated cells compared to control group. Results: The results showed that 1.5, 3, 6, and 12 mg/mL concentrations of ciprofloxacin have toxic effects on ADSCs (P < 0.0001) while in 0.5-150 µg/mL concentrations, there are no toxic effects on the cells. Furthermore, Oct-4 and Nanog expression in the nontoxic concentration of ciprofloxacin-treated cells was increased compared to the control group, which indicates the positive effect of ciprofloxacin on the growth of these cells. Conclusions: The present study shows that due to increased expression of Oct-4 and Nanog in 10 µg/mL ciprofloxacin-treated cells, the use of ciprofloxacin during the ADSCs culture period could provide suitable conditions for maintaining stem cell properties in these cells.