Spatial and temporal variations of oxygen environments affect the behaviors of various cells and are involved in physiological and pathological events. Our previous studies withDictyostelium discoideumas a model of cell motility have demonstrated that aerotaxis toward an oxygen-rich region occurs below 2% O2. However, while the aerotaxis ofDictyosteliumseems to be an effective strategy to search for what is essential for survival, the mechanism underlying this phenomenon is still largely unclear. One hypothesis is that an oxygen concentration gradient generates a secondary oxidative stress gradient that would direct cell migration towards higher oxygen concentration. Such mechanism was inferred but not fully demonstrated to explain the aerotaxis of human tumor cells. Here, we investigated the role on aerotaxis of flavohemoglobins, proteins that can both act as potential oxygen sensors and modulators of nitric oxide and oxidative stress. The migratory behaviors ofDictyosteliumcells were observed under both self-generated and imposed oxygen gradients. Furthermore, their changes by chemicals generating or preventing oxidative stress were tested. The trajectories of the cells were then analyzed through time-lapse phase-contrast microscopic images. The results indicate that both oxidative and nitrosative stresses are not involved in the aerotaxis ofDictyosteliumbut cause cytotoxic effects that are enhanced upon hypoxia.