Depression and anxiety are two of the most predominant mental disorders and leading causes of disability. The World Health Organization estimated that 3.8% of the world's population suffers from depression, and between 0.4% and 3.6% have an anxiety spectrum diagnosis. Psychedelics, particularly psilocybin, have been extensively studied as new and promising treatments, showing long-lasting positive results when combined with psychotherapy for patients with depression and anxiety, among other disorders. However, pre-clinical trials investigating the precise neurobiological mechanisms underlying these results are still scarce, especially with female subjects. Additionally, emerging evidence suggests that hormonal fluctuations may influence the drug's efficacy, potentially providing a protective effect. This study examined the behavioural effects of high doses of psilocybin on depression and anxiety-like measures in adult female rats. SERT knockout animals were tested with 4 or 8 mg/kg doses of psilocybin using the affective disorders test (ADT). A secondary analysis in a small group of animals assessed the impact of the oestrous cycle phase when the animals were injected. Results showed no robust effects on depression and anxiety-like measures with either dose. However, trends were observed in anxiety-like measures at 4 and 8 mg/kg. Evaluation indicated an interaction between treatment and the oestrous cycle phase, suggesting female hormones might protect against the drug's effects on depression-like measures.