We previously reported that physical inactivity (PI) induces cognitive decline and depressive states, which were ameliorated by regular exercise. However, the mechanism underlying the preventive effect of exercise remains unelucidated. Irisin has recently been identified as an exercise-inducible myokine that improves cognitive impairment. Plasma irisin levels increase during physical exercise; therefore, PI could lead to a decline in cognitive function by reducing plasma irisin. Therefore, this study aimed to examine whether irisin is associated with cognitive decline and mental deterioration altered by PI and exercise. The mice were housed for eight weeks in the PI cage, whose living space was one-sixth that of a standard cage. Simultaneously, the mice were subjected to regular exercise in the presence or absence of an irisin-neutralizing antibody. PI increased the epididymal fat mass without increasing body weight, muscle mass, or plasma corticosterone levels. Additionally, PI induced anxiety, depressive states, and a decline in working memory. In contrast, regular exercise after PI elevated irisin levels in plasma and increased fibronectin type III domain-containing 5 (FNDC5) and peroxisome proliferator-activated receptor gammacoactivator 1α expression in skeletal muscle. Regular exercise also increased hippocampal brain-derived neurotrophic factor (BDNF) expression and BrdU-positive cells, alleviating cognitive decline and mental deterioration induced by PI. The beneficial effects of exercise were compromised by the administration of an irisin-neutralizing antibody. Moreover, plasma irisin level was positively correlated with working memory, hippocampal BDNF levels, and hippocampal cell proliferation. These findings suggest that exercise-inducible irisin is critical for maintaining cognitive function in the PI state.