Working memory is a process for short-term active maintenance of information. Behavioral neurophysiological studies in monkeys have demonstrated that the dorsolateral prefrontal cortex (dlPFC) is a key cortical region for working memory. The medial prefrontal cortex (mPFC) in rats is a cortical area similar to the dlPFC in monkeys in terms of anatomical connections, and is also required for behavioral performance on working-memory tasks. However, it is still controversial regarding whether and how mPFC neurons encode working memory. In the present study, we trained rats on a two-choice spatial delayed alternation task in Y maze, a typical working memory task for rodents, and investigated neuronal activities in the mPFC when rats performed the task. Our results show that, (1) inactivation of the mPFC severely impaired the performance of rats on the task, consistent with previous studies showing the importance of the mPFC for working-memory tasks; (2) 93.7% mPFC cells (449 in 479) exhibited changes in spiking frequency that were temporally locked with the task events, some of which, including delay-related cells, were tuned by spatial information; (3) differential delay activities in individual mPFC cells appeared transiently and sequentially along the delay, especially during the early phase of the delay; (4) some mPFC cells showed no change in discharge frequency but exhibited differential synchronization in firing during the delay. The present results suggest that mPFC neurons in rats are involved in encoding working memory, via increasing firing frequency or synchronization.