High levels of small low density lipoprotein (LDL) particles are a major risk factor for cardiovascular morbidity and mortality. Both estrogens and smoking, with known anti-estrogenic effects, alter the atherogenic lipid profile. We tested for a role of interaction between smoking and estrogen receptor alpha gene (ESR1) variation in association with plasma concentration of atherogenic small LDL particles and LDL particle size. We studied 1727 unrelated subjects, 854 women and 873 men, mean age 51 years (SD 10), from the population-based Framingham Heart Study. After covariate adjustment, women who smoked and had the common ESR1 c.454-397 TT genotype (in 30% of women, T was present on both chromosomes at position 397 prior to the start of exon 2) had >1.7-fold higher levels of small LDL particles than women with the alternative genotypes (P-value for smoking-genotype interaction was 0.001). Similar results were obtained for three other ESR1 variants including c.454-351A > G, in the same linkage disequilibrium block. A similar substantial gender-specific result was also evident with a fifth variant, in a separate linkage disequilibrium block, in exon 4 (P = 0.003). Women who smoked and had specific, common ESR1 genotypes had a substantially higher plasma concentration of atherogenic small LDL particles. Significant results revealed a dose-dependent effect of smoking and were evident in both pre- and postmenopausal women. The reported association has the potential to explain the risks associated with estrogen use in certain women and a recent report of association between an ESR1 haplotype comprised of c.454-397 T and c.454-351 A alleles with increased myocardial infarction and ischaemic heart disease, independent of the standard, established cardiovascular risk factors.