Learning occurs in the context of existing memories. Encountering new information that relates to prior knowledge may trigger integration, whereby established memories are updated to incorporate new content. Here, we provide a critical test of recent theories suggesting hippocampal (HPC) and medial prefrontal (MPFC) involvement in integration, both during and immediately following encoding. Human participants with established memories for a set of initial (AB) associations underwent fMRI scanning during passive rest and encoding of new related (BC) and unrelated (XY) pairs. We show that HPC–MPFC functional coupling during learning was more predictive of trial-by-trial memory for associations related to prior knowledge relative to unrelated associations. Moreover, the degree to which HPC–MPFC functional coupling was enhanced following overlapping encoding was related to memory integration behavior across participants. We observed a dissociation between anterior and posterior MPFC, with integration signatures during post-encoding rest specifically in the posterior subregion. These results highlight the persistence of integration signatures into post-encoding periods, indicating continued processing of interrelated memories during rest. We also interrogated the coherence of white matter tracts to assess the hypothesis that integration behavior would be related to the integrity of the underlying anatomical pathways. Consistent with our predictions, more coherent HPC–MPFC white matter structure was associated with better performance across participants. This HPC–MPFC circuit also interacted with content-sensitive visual cortex during learning and rest, consistent with reinstatement of prior knowledge to enable updating. These results show that the HPC–MPFC circuit supports on- and offline integration of new content into memory.