In this study, mixed electron donors of Mn(II) and Fe(II) were tested for their effects on autotrophic denitrification. The denitrification performance based on various Mn(II)/Fe(II) molar ratios was assessed via batch experiments conducted under different conditions of temperature, pH, and electron donor/electron acceptor molar ratios. Complete nitrate removal was found at high Mn(II)/Fe(II) molar ratios, 30 degrees C, pH 7.0, and an electron donor/electron acceptor molar ratio of 3.00. Furthermore, we assessed the expression of nirS through amplification approaches and determined N-2 levels by gas chromatography. Our results demonstrated that mixed electron donors are efficient for autotrophic denitrification, and strain SZF15 is a suitable candidate for the simultaneous removal of nitrogen, Fe(II), and Mn(II) in groundwater treatment. The present work will benefit the development of autotrophic denitrification technology.