Aromatase catalyzes the conversion of testosterone to estradiol and is involved in the physiological effects of sex hormones on brain function. Animal experiments have shown that the aromatase inhibitor, letrozole, can induce anxiety in young ovariectomized females that are used as a model of aging. Whether or not these effects would be similar in intact middle‐aged animals is unknown. The aim of our study was to analyze the effects of letrozole on anxiety in middle‐aged rats of both sexes. Fifteen month old male and female rats were treated daily with either letrozole or vehicle for 2 weeks. The elevated plus maze was used to test anxiety‐like behaviour. Sex differences were found not only in plasma concentrations of testosterone but also in the effects of letrozole treatment on plasma testosterone (P<.05). The interaction between sex and treatment was also proven in locomotor activity (P<.05) and time spent in the open arms of the elevated plus maze (P<.05). Letrozole‐treated male rats spent 95% less time in the open arms of the elevated plus maze than the control rats did (P<.05) suggesting an anxiogenic effect of aromatase inhibition. This difference was not found between letrozole‐treated and vehicle‐treated females. In contrast to previous experiments on young animals, letrozole seems to induce anxiety in male but not in female middle‐aged rats. This sex‐specific effect might be related to sex differences of oestrogen and androgen signalling in aging brains. These results should be taken into account in clinical applications of letrozole, especially in men.