[Background] With increasing numbers of drug resistant microorganisms and drained antibiotics sources, research on bacterial drug resistance mechanisms has risen to be a most urgent scientific topic. [Objective] To understand how endogenous cysteine affects ROS levels in Escherichia coli by promoting the Fenton reaction. [Methods] The effect of cysteine on the antibiotic resistance of E. coli was investigated by controlling the concentration of exogenous cystine and the concentration of antibiotics (gentamicin, ampicillin, norfloxacin) under poor sulfur conditions. [Results] Low concentrations of cysteine increased drug resistance of E. coli to all of the antibiotics used in this study. The regulation on Fur, CysB or SOS by influx of cystine was also confirmed using RNA-Seq. An analysis of sulfur efflux by LC-MS showed that excess cysteine was excluded immediately. At certain concentrations of antibiotics, the efflux pump AlaE exhibited favorable protection on cells. [Conclusion] Drug resistance of E. coli to gentamicin, ampicillin, norfloxacin, antibiotics with different mechanisms of action, was affected by endogenous cysteine levels. Our current work provides new theoretical information on bacterial drug-resistance mechanisms.