Hippocampal theta activity is important for the acquisition of spatial information and is strongly influenced and regulated by extra-hippocampal inputs from the synchronising ascending system (SAS), which includes the supramammillary nucleus (SUMn) and the posterior hypothalamic nucleus (PHn). Together these nuclei play an important role in controlling the frequency encoding of theta activity and are innervated by serotonin synapses, which also regulate theta activity and learning abilities. The participation of the SUMn in place learning and modulation of hippocampal theta activity were recently shown; thus, we questioned whether serotonin acting on SUMn/PHn could modulate place learning ability and concurrent hippocampal theta activity. The serotonergic terminals of the SUMn/PHn in rats were lesioned through 5,7-dihydroxytryptamine (5,7-DHT) infusion, and hippocampal theta activity during the Morris water maze test was recorded. Rats in the vehicle group learned the task efficiently and showed learning-related theta changes in the CA1 and dentate gyrus regions throughout the training. The 5-HT-depleted rats were deficient in the Morris water maze task and showed theta activity in the CA1 and dentate gyrus that were unrelated to the processing of learning. We conclude that serotonin can regulate the hippocampal theta activity acting on the SUMn/PHn relay of the SAS and that the influence of 5-HT in these nuclei is required for the learning-related changes in hippocampal theta activity that underlie the successful resolution of the Morris water maze task.