Increasing evidence indicates an important role of steroid-binding proteins in endocrine functions, including hypothalamic–pituitary–adrenal (HPA) axis activity and regulation, as they influence bioavailability, local delivery, and cellular signal transduction of steroid hormones. In the plasma, glucocorticoids (GCs) are mainly bound to the corticosteroid-binding globulin (CBG) and to a lesser extend to albumin. Plasma CBG levels are therefore involved in the adaptive stress response, as they determine the concentration of free, biologically active GCs. In this study, we investigated whether male mice with a genetic predisposition for high-reactivity (HR), intermediate-reactivity (IR), or low-reactivity (LR) stress-induced corticosterone (CORT) secretion present different levels of free CORT and CORT-binding proteins, basally and in response to stressors of different intensity. Our results suggest a fine control interaction between plasma CBG expression and stress-induced CORT release. Although plasma CBG levels, and therefore CBG binding capacity, were higher in HR animals, CORT secretion overloaded the CBG buffering function in response to stressors, resulting in clearly higher free CORT levels in HR compared with IR and LR mice (HR>IR>LR), resembling the pattern of total CORT increase in all three lines. Both stressors, restraint or forced swimming, did not evoke fast CBG release from the liver into the bloodstream and therefore CBG binding capacity was not altered in our three mouse lines. Thus, we confirm CBG functions in maintaining a dynamic equilibrium between CBG-bound and unbound CORT, but could not verify its role in delaying the rise of plasma free CORT immediately after stress exposure.