Early life stress (ELS) plays a crucial role in shaping adult behaviour and has been implicated as a risk factor in several mental disorders, including schizophrenia. The α5-subunit of the nicotinic acetylcholine receptor (nAChR) has also been associated with disorders such as addiction and schizophrenia. However, the combined effect of ELS and α5-nAChR function remains unexplored. Here, we investigated this interaction by subjecting male and female mice lacking α5-nAChRs (ACNA5) and wild-type (WT) mice to maternal separation (MS) during postnatal days 2-16 (3 hours daily). The animals were examined in early adulthood with a battery of behavioural tasks assessing locomotion, anxiety, and memory. In the open-field test, ACNA5 mice exhibited greater activity than WT, with MS increasing locomotion only in male animals of both genotypes. In the elevated plus maze, female ACNA5 mice showed increased anxiety compared to WT, while MS had no significant impact on anxiety levels in any group. In the novel-object-location task, male ACNA5 mice showed impaired memory, while MS was associated with a tendency for reduced performance in WT males and ACNA5 females. Lastly, in the novel-object-recognition test, memory was better in ACNA5 males, but worse in ACNA5 females, while MS improved performance in all groups except ACNA5 male mice. Taken together, our results reveal that the effects of MS on mouse behaviour depend on both the sex and the genotype of the animals, and that deletion of α5-nAChRs can either protect from ELS or enhance its impact, with male ACNA5 mice being the most susceptible.