A common treatment for anxiety disorders is chronic administration of selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine. Recent data suggest that SSRIs modulate fear responses after conditioned fear extinction and that gonadal hormones influence fear extinction. In this study we investigated the influence of sex and the estrous cycle on the effects of acute (experiment 1) and chronic (experiment 2) fluoxetine treatment on fear extinction. In experiment 1, rats received tone-footshock pairings during day 1. On day 2, rats received either fluoxetine (10 mg/kg in 0.5 mL) or vehicle prior to extinction learning. On day 3, extinction memory was assessed during extinction recall. In experiment 2, rats were exposed to a similar behavioral protocol, except that fluoxetine and vehicle were administered for 14 consecutives days after conditioning (days 2–15). Extinction learning and extinction recall occurred on days 15 and 16, respectively. Acute administration of fluoxetine increased fear responses equally in males and females during extinction learning and extinction recall. Chronic administration of fluoxetine reduced fear responses during extinction learning and extinction recall in female but not in male rats and this effect seems to be modulated by the estrous cycle. The SSRI-induced reduction of freezing during extinction learning and recall suggest a general anxiolytic effect of the drug treatment rather than a specific effect on extinction learning per se. Our data show evidence of sex-specific anxiolytic effects of 14-day treatment of fluoxetine while the acute anxiogenic effect of SSRI seems independent of sex effects.