Pattern separation (PS) describes the process by which the brain discriminates similar stimuli from previously encoded stimuli. This fundamental process requires the intact processing by specific subfields in the hippocampus and can be examined using mnemonic discrimination tasks. Previous studies reported different patterns for younger and older individuals between mnemonic discrimination performance and hippocampal subfield activation. Here, we investigated the relationship between the lure discrimination index (LDI) and hippocampal subfield volume and activity across the adult lifespan (20-70 years old). Using ultra-high field functional and structural magnetic resonance imaging at 7 T, we found that lower DG volume and higher CA3 activation was associated with worse LDI performance in individuals (>60 years), suggesting that this higher activation may be an indication of aberrant neurodegenerative-related processes. In fact, higher activation in the CA1 and DG was associated with lower volumes in these subfields. For individuals around 40-50 years old, we observed that greater left and right DG volume, and greater activity in the CA3 was associated with lower LDI performance. Taken together, these results suggest that the relationship between memory and hippocampal subfield structure or function varies nonlinearly and possibly reciprocally with age, with midlife being a critically vulnerable period in life. A decline in cognitive functions is part of normal ageing 1 and for older adults, memory problems, in particular in episodic memory, are considered among the most worrisome. The hippocampus is known to play a central role in episodic memory 2,3. The hippocampus is not a homogeneous structure, different subfields are involved in different memory processes 4. This has in particular been shown for pattern separation and completion, the most extensively investigated memory processes in the context of distinct hippocampal subfield affinities. Pattern separation, the ability to form distinct, non-overlapping representations from similar or overlapping inputs has been shown to rely on the dentate gyrus (DG) and Cornu Ammonis (CA3) regions in human studies 5-10. A distinction is made between behavioural pattern separation of objects and of spatial locations 4. While the downstream pathways for spatial pattern separation involve medial entorhinal cortices, object pattern separation engages the lateral entorhinal cortices. In the hippocampal subfields CA3/DG the distinction between object and spatial pattern separation is no longer present 11. Both variants of pattern separation (object and spatial) are similarly affected by age. Age-related changes in pattern separation have been documented extensively in animal 12 and human studies 4,13. Atrophy of the DG in rodent models of ageing correlated with discrimination deficits 14. Behavioural work demonstrated a monotonic decline in discrimination abilities starting in the fourth decade in