In human lysozyme (hLYZ) production by Pichia pastoris, the glycerol fed-batch phase was generally implemented under the environment of "oxygen sufficient-glycerol limited" to achieve high cell-density cultivation during the cell growth phase. However, the structural and functional components in P. pastoris cells were irreversible damaged with more and more reactive oxygen species (ROS) accumulation when cells were exposed to the oxygen sufficient environments for long time, leading to a failure of hLYZ expression. In this study, a novel periodic glycerol and dissolved oxygen concentration (DO) control strategy was proposed to solve these problems. This strategy periodically switched the cultivation environments from "oxygen sufficient-glycerol limited" to "oxygen limited-glycerol sufficient" for 5 cycles. When using this strategy: (1) the highest dry cell weight (DCW) of 143.02 g-DCW/L and the lowest distribution of glycerol towards to cell maintenance (0.0400 1/h) were achieved during the glycerol feeding phase by maintaining ROS levels below 48.39 Fluorescence intensity/g-DCW; (2) the adaption time of P. pastoris cells to methanol induction environments was shortened for about 50%; (3) P. pastoris cell metabolic activities reflected by the activities of alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and methanol consumption rate, etc., in the successive induction phase were largely enhanced; (4) hLYZ activity reached the highest level of 2.45 × 10 IU/mL, which was about 2-fold than that obtained with the strategy of "oxygen sufficient-glycerol limited," when the same methanol induction strategy was adopted. KEY POINTS: • A novel periodic glycerol feeding strategy proposed/used for P. pastoris cell growth. • Higher cell density was obtained by controlling ROS at low level via this strategy. • The highest hLYZ activity was achieved when initiating induction at higher cell density.