Acetonitrile contamination in an operating cell and performance recovery are investigated in detail by a series of electrochemical characterizations, including constant current operation, electrochemical impedance spectroscopy, cyclic voltammetry, linear scanning voltammetry and chronoamperometry. We demonstrate that 20 ppm of acetonitrile in the air stream causes an approximate 40% reduction in cell performance at 1 A cm−2; however, the neat air operation completely restores the single cell. Cell performance degradation and recovery display two distinguishable periods that correspond to the effect of catalyst activity and electrolyte proton conductivity, respectively. The potential effect on acetonitrile contamination is also studied in a non-operating H2/N2 cell. The results indicate that low and high potentials cause a larger proton conductivity effect than a medium potential. Two recovery methods were conducted to restore cell performance after contamination in N2 at different potentials. The neat air operation completely mitigated the poisoning effect in approximately 5 h regardless of contamination history; however, the N2 purge and subsequent potential scans, even scanning up to 1.5 V vs hydrogen reference electrode, did not affect the poisoned cell. The liquid water generated in catalyst layers may play an important role during the recovery from acetonitrile contamination in a proton exchange membrane fuel cell.