The ingestion of the beverage Ayahuasca usually occurs in religious ceremonies that are performed during the night leading to sleep deprivation. The purpose of the present study was to characterize the acute effects of Ayahuasca upon the sexual response of sleep deprived male rats. One group of sexually experi- enced male Wistar rats were submitted to a paradoxical sleep deprivation (PSD) protocol for 96 h, while another group spent the same amount of time in the home cage (CTRL). After this period, either saline or Ayahuasca drink (250, 500 and 1000 g mL−1 ) was administered by gavage and sexual behavior and hormonal concentrations were measured. Ayahuasca alone significantly decreased sexual performance at all doses. However, in sleep deprived rats, the lower dose increased sexual performance while the intermediate dose produced a detrimental effect on sexual response compared to the CTRL rats at the same dose. Regarding the hormonal analyses, a lower testosterone concentration was observed in sleep- deprived saline rats in relation to the CTRL group. Progesterone was significantly lower only in PSD rats at the dose 500 g mL−1 compared with CTRL-500 g mL−1 group. Corticosterone was unchanged among the groups evaluated. Our results suggest that Ayahuasca intake markedly impaired sexual performance alone, but, when combined with sleep deprivation, had significant, but heterogeneous, effects on male sexual response.