Background Estrogen is involved in the pathogenesis of breast and gynecological cancers. Regular use of aspirin reduces estrogen levels. The present study aimed to evaluate the effect of aspirin on estrogen levels in postmenopausal women. Methods This double-blind, placebo-controlled parallel-group trial was conducted on postmenopausal women referred to an outpatient clinic at a women’s hospital in Tehran. Volunteers were randomly assigned to receive aspirin 100 mg/day or placebo for 6 weeks. Estradiol, sex hormone-binding globulin (SHBG), and testosterone levels at baseline and at the end of the intervention were measured by ELISA. Data were analyzed using SPSS 20, Kolmogorov–Smirnov test, independent samples t-test, and Mann–Whitney U test. Results Twenty-seven and 28 participants were finally analyzed in the aspirin and placebo groups, respectively. There was no significant difference between the two groups in body mass index (BMI), age, or menopausal years. There was a statistically significant difference (p = 0.002) in the amount of  change in estradiol levels of the intervention group (median=− 3.5 pg/ml) compared to the control group (median=1.5 pg/ml). In contrast, there were no significant differences between the two groups regarding testosterone and SHBG levels (p = 0.58, p = 0.32). Conclusions Since low doses of aspirin may decrease estradiol levels, it could be considered a promising adjunctive therapeutic candidate in postmenopausal women to decrease BC incidence. However, further studies with larger sample sizes, measurements of estrogen levels and its related compounds in different time points accompanied by long-term follow-ups are needed to better elucidate the potential mechanisms by which nonsteroidal anti-inflammatory drugs (NSAIDs) negatively affect breast cancer. Trial registration IRCT201012195397N1. Date of first registration: 03/01/2011.