We investigated the associations between smoking status and metabolic risk factors and sex hormones in polycystic ovary syndrome (PCOS). The study was designed as a retrospective trans-sectional study including 650 white premenopausal women with the diagnoses hirsutism or PCOS divided according to smoking status: non-smokers (NS-PCOS = 390) and smokers (S-PCOS = 260). One hundred and nineteen healthy women were studied as controls (NS-Control = 105, S-Control = 14). Patients and controls underwent clinical evaluation, hormone analyses, transvaginal ultrasound, oral glucose tolerance tests (OGTT) and adrenocorticotropic hormone (ACTH) tests. S-PCOS has significantly higher fasting lipid profile and 17-hydroxyprogesterone levels (basal and ACTH-stimulated) than NS-PCOS patients, whereas prolactin levels were decreased. No significant differences were found in body composition and measures of insulin resistance between NS-PCOS and S-PCOS. PCO was more prevalent in NS-PCOS patients. During multiple regression analyses, smoking was positively associated with 17-hydroxyprogesterone (17OHP) and cholesterol, triglycerides and low-density lipoprotein and inversely associated with prolactin and high-density lipoprotein. We concluded that smoking was associated with increased adrenal responsiveness, decreased prolactin levels and a more adverse lipid profile in PCOS patients, whereas smoking was unassociated with body composition and insulin resistance. Smoking may be associated with the prevalence of individual Rotterdam criteria.