Background Prenatal tobacco smoke exposure may be associated with low maternal folate levels that increase the risk of adverse infant and child health outcomes by reducing folate availability during fetal development. Methods Using data from the HOME Study, we examined the relationship between secondhand or active tobacco smoke exposure and whole blood folate concentrations in pregnant women from Cincinnati, OH (n=362) at approximately 16 weeks gestation. We used multivariable linear regression to examine the association between continuous or categorical serum cotinine levels and whole blood folate levels, adjusting for sociodemographic, dietary, and perinatal variables. Results After adjustment for potential confounders, an interquartile range increase in serum cotinine concentration (0.012 to 0.224 ng/mL) was suggestively associated with decreased whole blood folate levels (β:−23 nmol/L; 95% CI:−49, 3, p-value=0.08). Compared to unexposed women, reductions in mean whole blood folate were observed among active smokers (β:−94, 95% CI:−195, 6 nmol/L, p-value=0.40); smaller reductions were observed among women with secondhand exposure (β:−26; CI:−84, 32 nmol/L, p-value=0.07). Conclusions Consistent with prior studies, active smoking was associated with reduced whole blood folate levels among these pregnant women. Secondhand tobacco smoke exposures were associated with small and imprecise reductions in whole blood folate levels.