Animal models of depression seldom test females, even though women are twice as likely as men to suffer from major depressive disorder. Since female mice are sensitive to social isolation, we tested a separation-based model of depression in three experiments. In experiment 1 female C57BL/6J mice were housed in three conditions: isolated (housed individually from 8 weeks of age), separated (housed in groups and then separated and housed individually at 23 weeks of age) and grouped (housed in groups from 8 weeks of age). At 24 weeks of age, there was a significant increase in weight and in immobility in individually housed mice in the forced swim test (FST) and tail suspension test (TST), a reduction in transitions in the L/D box, a reduced startle response and reduced prepulse inhibition, but no differences in cued or context fear conditioning. Experiment 2 showed that fluoxetine treatment administered via drinking water attenuated depressive-like behaviour in the FST and TST in individually housed female C57BL/6J mice, but had no effect on anxiety-like behaviour. Experiment 3 found that group-housed females had higher baseline corticosterone (CORT) levels than isolated females and fluoxetine had no effect on CORT levels. Thus, separation from group housing is a reliable and valid method for inducing depression-like behaviour in female mice. This procedure is both versatile, allowing for the study of genetic and environmental interactions, and accessible, making it useful for studying depression and testing new drugs for its treatment.