Diverse neural processes have been proposed as the neural basis of working memory. To investigate whether the medial prefrontal cortex (mPFC) relies on different neural processes to mediate working memory depending on the predictability of delay duration, we examined mPFC neural activity in mice performing a delayed response task with fixed (4 s) or random (between 1-7 s) delay durations. mPFC neural activity was strongly influenced by the predictability of delay duration. Nevertheless, mPFC neurons seldom showed persistent activity spanning the entire delay period and instead showed dynamically-changing delay-period activity under both the fixed- and random-delay conditions. mPFC neurons conveyed higher working memory information under the random- than fixed-delay conditions, possibly due to a higher demand for stable working memory maintenance. Our results support the possibility that the mPFC relies on dynamically-changing neuronal population activity to maintain working memory regardless of the predictability of delay duration.