Persistent activity is thought to mediate working memory during behavior. Can it also occur during sleep? We found that the membrane potential of medial entorhinal cortex layer III (MECIII) neurons, a gateway between neocortex and hippocampus, showed spontaneous, stochastic persistent activity in vivo in mice during Up-Down state oscillations (UDS). This persistent activity was locked to the neocortical Up states with a short delay, but persisted over several cortical UDS cycles. Lateral entorhinal neurons did not show substantial persistence, and current injections similar to those used in vitro failed to elicit persistence in vivo, implicating network mechanisms. Hippocampal CA1 neurons' spiking activity was reduced during neocortical Up states, but was increased during MECIII persistent states. These results provide, to the best of our knowledge, the first direct evidence for persistent activity in MECIII neurons in vivo and reveal its contribution to cortico-hippocampal interaction that could be involved in working memory and learning of long behavioral sequences during behavior, and memory consolidation during sleep.