BACKGROUND Some mood disorders, such as major depressive disorder, are more prevalent in women than in men. However, historically preclinical studies in rodents have a lower inclusion rate of females than males, possibly due to the fact that behavior can be affected by the estrous cycle. Several studies have demonstrated that chronic antidepressant treatment can decrease anxiety-associated behaviors and increase adult hippocampal neurogenesis in male rodents. OBJECTIVE Very few studies have looked at the effects of antidepressants on behavior and neurogenesis across the estrous cycle in naturally cycling female rodents. METHODS Here, we analyze the effects of chronic treatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine (Prozac) on behavior and adult hippocampal neurogenesis in naturally cycling C57BL/6J females across all four phases of the estrous cycle. RESULTS In naturally cycling C57BL/6J females, fluoxetine decreases negative valence behaviors associated with anxiety in the elevated plus maze and novelty-suppressed feeding task, reduces immobility time in forced swim test, and increases adult hippocampal neurogenesis. Interestingly, the effects of fluoxetine on several negative valence behavior and adult hippocampal neurogenesis measures were mainly found within the estrus and diestrus phases of the estrous cycle. CONCLUSIONS Taken together, these data are the first to illustrate the effects of fluoxetine on behavior and adult hippocampal neurogenesis across all four phases of the murine estrous cycle.