Several cortical and subcortical circuits have been implicated in object location memory and navigation. Uncertainty remains, however, about which neural circuits are involved in the distinct processes of encoding and retrieval during active navigation through three-dimensional space. We used functional magnetic resonance imaging (fMRI) to measure neural responses as participants learned the location of a single target object relative to a small set of landmarks. Following a delay, the target was removed and participants were required to navigate back to its original position. The relative and absolute locations of landmarks and the target object were changed on every trial, so that participants had to learn a novel arrangement for each spatial scene. At encoding, greater activity within the right hippocampus and the parahippocampal gyrus bilaterally predicted more accurate navigation to the hidden target object in the retrieval phase. By contrast, during the retrieval phase, more accurate performance was associated with increased activity in the left hippocampus and the striatum bilaterally. Dividing participants into good and poor navigators, based upon behavioural performance, revealed greater striatal activity in good navigators during retrieval, perhaps reflecting superior procedural learning in these individuals. By contrast, the poor navigators showed stronger left hippocampal activity, suggesting reliance on a less effective verbal or symbolic code by this group. Our findings suggest separate neural substrates for the encoding and retrieval stages of object location memory during active navigation, which are further modulated by participants' overall navigational ability.