We examined the effects of aging and cognitive fatigue on working memory (WM) related brain activation using functional magnetic resonance imaging. Age-related differences were investigated in 13 young and 16 middle-aged male school teachers. Cognitive fatigue was induced by sustained performance on cognitively demanding tasks (compared to a control condition). Results showed a main effect of age on left dorsolateral prefrontal and superior parietal cortex activation during WM encoding; greater activation was evident in middle-aged than young adults regardless of WM load or fatigue condition. An interaction effect was found in the dorsomedial prefrontal cortex (DMPFC); WM load-dependent activation was elevated in middle-aged compared to young in the control condition, but did not differ in the fatigue condition due to a reduction in activation in middle-aged in contrast to an increase in activation in the young group. These findings demonstrate age-related activation differences and differential effects of fatigue on activation in young and middle-aged adults.