Abstract Objective Current literature suggests there may be a relationship between sex hormones, which dramatically increase during pregnancy, and nicotine use behaviors. We hypothesized that higher progesterone and progesterone:estradiol ratio (P/E₂) would be associated with less smoking-related symptomatology (SRS), better mood and fewer cigarettes smoked per day (CPD) during ad libitum smoking and following overnight abstinence in pregnant women. Associations between SRS, mood, smoking behavior and sex hormones were estimated using multiple linear regression with adjustment for CPD and pregnancy trimester. Results There were 35 second trimester and 42 third trimester participants. Participants mean age was 26.2 (SD: 4.1), they smoked 11.3 CPD (SD: 4.4) and the mean nicotine dependence score was 4.94 (SD: 1.98). There were no statistically significant associations between progesterone levels, estradiol levels, or the P/E₂ ratio and SRS or mood measures during ad libitum smoking or following overnight abstinence in this sample of pregnant women. Similarly, there were no associations between sex hormone levels and number of CPD smoked during the ad libitum period. Contrary to our hypothesis, we found no significant associations between sex hormones and SRS, mood or smoking behavior in this sample of pregnant women. Trial registration ClinicalTrials.gov (NCT01811225), December 6, 2012