Introduction: This study aimed to examine the association of smoking status with homocysteine levels and to determine whether the association is modified by oestradiol or cholesterol. Methods: Data (N = 4580) were obtained from National Health and Nutrition Examination Survey 2003–2004 with analysis done in 2018 on adults aged ≥20 years. The outcome was homocysteine; smoking status was the exposure variable and categorized as current, former or never smoker. Generalized linear models were used to examine the associations between smoking status and homocysteine levels, while assessing the impact of oestradiol and cholesterol. Results: After adjusting for age, sex, ethnicity, education and income level, homocysteine levels did differ by smoking status ((current smokers versus never smokers: β: 0.18 CI: 0.00, 0.36), (former smokers: β: 0.10 CI: –0.09, 0.28)). The addition of oestradiol as an interaction term in adjusted models was associated with a 16.6% increase in homocysteine levels when compared to models without the interaction term. Oestradiol but not cholesterol did moderate the association between smoking status and homocysteine levels. Discussion and conclusions: Homocysteine levels did differ across smoking status after adjusting for confounders. Oestradiol did moderate the relationship between homocysteine and smoking status.