Cigarette smoking has been implicated in reproductive outcomes including delayed conception, but mechanisms underlying these associations remain unclear. One potential mechanism is the effect of cigarette smoking on reproductive hormones; however, studies evaluating associations between smoking and hormone levels are complicated by variability of hormones and timing of specimen collection. We evaluated smoking among women participating in the BioCycle Study, a longitudinal study of menstrual cycle function in healthy, premenopausal, regularly menstruating women (n = 259). Fertility monitors were used to help guide timing of specimen collection. Serum levels of oestradiol, progesterone, follicle-stimulating hormone (FSH), luteinising hormone (LH) and total sex-hormone binding globulin (SHBG) across phases of the menstrual cycle were compared between smokers and non-smokers. We observed statistically significant phase-specific differences in hormone levels between smokers and non-smokers. Compared with non-smokers, smokers had higher levels of FSH in the early follicular phase and higher LH at menses after adjusting for potential confounding factors of age, race, body mass index, parity, vigorous exercise, and alcohol and caffeine intake through inverse probability of treatment weights. No statistically significant differences were observed for oestradiol, progesterone or SHBG. These phase-specific differences in levels of LH and FSH in healthy, regularly menstruating women who are current smokers compared with non-smokers reflect one mechanism by which smoking may influence fertility and reproductive health.