With the imminence of global climate changes that affect the temperature and the rainfall uniformity, it is growing the concern about the adaptation of crops to the water deficit. Thus, the objective of this study was to evaluate alterations in physiological and enzymatic mechanisms during the germination process of sesame seeds under different water availability. To simulate the water restriction we used PEG6000, a high molecular weight molecule that does not penetrate the seed structure but allows different osmotic potentials. The treatments were -0.1, -0.2, and -0.3 MPa, and the control. Germination, first-count germination, germination velocity index, and length and dry mass of the hypocotyl and radicle were performed. The seeds were weighed before and after treatments every 3 h. After each weighing, 100 seeds were taken for analysis of the enzymes alcohol dehydrogenase (ADH), malate dehydrogenase, esterase, catalase (CAT), superoxide dismutase (SOD), isocitrate lyase (ICL), and glutamate dehydrogenase (GTDH). The statistical design was completely randomized with five replications. PEG6000 prolonged ADH activity during the beginning of germination, maintaining the anaerobic metabolism for longer. Subsequently, their activity was reduced, as well as ICL, favoring the deterioration of the seeds that take the time to germinate. Behavior was evidenced by the appearance of SOD, CAT, and GTDH isoforms after 24 h of imbibition when water restriction was imposed. Therefore, the PEG600 is efficient in simulating water deficit conditions in future scenarios of climate change, offering impotent information regarding the germination behavior of the plants under these conditions.