Visuospatial working memory is one of the important cognitive processes and makes it possible to maintain and manipulate the visual information stored in brain systems. In this study, effects of task difficulty and two-week training of visuospatial working memory task on brain activity and its neural mechanisms were investigated. We used visuospatial working memory task with 10 or 30 visual targets displayed on a screen. Each target was a numbered white square and subjects were instructed to click the targets sequentially. Brain activity during performance of the task was measured with functional magnetic resonance imaging (fMRI), before and after two weeks task training. Four subjects practiced the task with 30 visual targets 15 times a day for two weeks, and another four subjects were not practiced. Statistical probability mapping revealed high activity in prefrontal cortex during performance of the task, especially in Brodmann area 10 and 46, which are commonly related to working memory processes. Higher and larger activation was observed for the task with 30 targets than that with 10 targets before training. After training, activated level and area of BA10 and 46 was increased and expanded in tasks with 10.