Much research has indicated that the mitogen-activated protein kinase (MAPK)-cAMP response element-binding protein (CREB) signal transduction pathway is involved in the pathophysiological mechanism of depression. But as to the question of which MAPKs are more relevant to stress effects, there is no definite answer. In the present study, 32 male Sprague–Dawley rats were divided into chronic unpredictable stress (CUS) and control groups, with 16 rats in each group. The CUS rats were exposed to 21-day chronic unpredictable stressors, and the controls were stress-free. After stress, 16 rats (8 in each group) were tested for spatial memory using Morris Water Maze, and 16 rats (8 from each group) were decapitated for detection of the three most extensively studied subgroups of MAPKs, ERK1/2, JNK and P38, and CREB in the hippocampus. The results showed that there was no statistical difference in the body weight between the two groups. The CUS rats showed impaired spatial memory in MWM. Western blot of hippocampus showed that CUS significantly decreased pCREB and pJNK levels, but there was no statistical difference between two groups in CREB, ERK1/2, pERK1/2, P38, pP38 and JNK levels. Immunohistochemistry showed that the reduced pCREB occurred in the dentate gyrus, not in the hippocampus proper. In conclusion, this study highlights that the JNK-CREB pathway, not the P38-CREB or ERK1/2-CREB pathway, in the hippocampus played an important role in the 21-day-CUS, and that the impaired spatial memory acquisition in the CUS rats can be restored to the level comparable to the pre-stressed state.