Objective:To investigate the most effective dosage and optimal time window of Plasmocin in eliminating mycoplasma infection,and detect whether the drug exert negative effects on human pluriotent stem cell(hPSC) culture. Methods:hPSCS were treated with 25.0,12.5,8.5,5.0 μg/mL of Plasmocin. The efficiency for removing mycoplasma infection was compared by PCR. Directly differentiation of hPSC to forebrain neurons after rescued. Results:All of the concentrations for Plasmocin eradicated mycoplasma infection. However,the highest dosage (25.0 μg/mL)lead to toxic effects and the lowest dosage(5.0 μg/mL)required longer treatment time. The 12.5 μg/mL group had adverse effects on cell growth but the 8.5 μg/mL group contributed to hPSCs maintenance. After mycoplasma elimination,hPSC efficiently differentiated to forebrain neurons. Furthermore,after 9 months post-Plasmocin treatment,mycoplasam was undetected on hPSC. Conclusion:One week treatment with 8.5 μg/mL Plasmocin completely eradicated mycoplasma contamination without affecting hPSC maintenance and differentiation.