The effect of microchip pulsed electric field (MPEF) treatment on lethal and sublethal injury of Pichia rhodanensis (P. rhodanensis) were employed under 100-500 V for 20-100 pulses and the underlying mechanism of MPEF treatment was investigated as well. A 6.48 log10 reduction of P. rhodanensis was achieved at 500V for 80 pulse. The fluorescent staining with Propidium Iodide (PI) verified that the rate of sublethal injury cells maximum up to 27.2% under 200 V. MPEF can cause the damage of cell morphology and ultrastructure, meanwhile causing a decrease in cellular enzymes, antioxidant enzyme activity and cell membrane fluidity. The leakage of intracellular compounds (protein, nucleic acid, K+, Mg2+) and Ca2+-ATPase gradually increased as the growth of voltage, especially the proportion of protein in the supernatants increased from 2.0% to 26.4%. Flow cytometry analysis showed that MPEF has significant effect on membrane potential, but no obvious influence on non-specific esterase. MPEF can cause the changing of the secondary structure of protein, at the same time, double helix structure of DNA became loose and unwinding. These results provide a theoretical guidance for the widespread using of MPEF technology in the application of a non-thermal processing technique for food.