<span class=”paragraphSection”><div class=”boxTitle”>Abstract</div><div class=”boxTitle”>Introduction:</div>Cigarette smoking is inversely associated with DNA methylation of the aryl hydrocarbon receptor repressor (<span style=”font-style:italic;”>AHRR</span>; cg05575921). However, the association between secondhand tobacco smoke (SHS) exposure and <span style=”font-style:italic;”>AHRR</span> methylation is unknown.<div class=”boxTitle”>Methods:</div>DNA methylation of <span style=”font-style:italic;”>AHRR</span> cg05575921 in CD14+ monocyte samples, from 495 never-smokers and 411 former smokers (having quit smoking ≥15 years) from the Multi-Ethnic Study of Atherosclerosis (MESA), was cross-sectionally compared with concomitantly ascertained self-reported SHS exposure, urine cotinine concentrations, and estimates of air pollutants at participants’ homes. Linear regression was used to test for associations, and covariates included age, sex, race, education, study site, and previous smoking exposure (smoking status, time since quitting, and pack-years).<div class=”boxTitle”>Results:</div>Recent indoor SHS exposure (hours per week) was inversely associated with cg05575921 methylation (β ± <span style=”font-style:italic;”>SE</span> = −0.009 ± 0.003, <span style=”font-style:italic;”>p</span> = .007). The inverse effect direction was consistent (but did not reach significance) in the majority of stratified analyses (by smoking status, sex, and race). Categorical analysis revealed high levels of recent SHS exposure (≥10 hours per week) inversely associated with cg05575921 methylation (β ± <span style=”font-style:italic;”>SE</span> = −0.28 ± 0.09, <span style=”font-style:italic;”>p</span> = .003), which remained significant (<span style=”font-style:italic;”>p</span> < .05) in the majority of stratified analyses. cg05575921 methylation did not significantly (<span style=”font-style:italic;”>p</span> < .05) associate with low to moderate levels of recent SHS exposure (1–9 hours per week), urine cotinine concentrations, years spent living with people smoking, years spent indoors (not at home) with people smoking, or estimated levels of air pollutants.<div class=”boxTitle”>Conclusions:</div>High levels of recent indoor SHS exposure may be inversely associated with DNA methylation of <span style=”font-style:italic;”>AHRR</span> in human monocytes.<div class=”boxTitle”>Implications:</div>DNA methylation is a biochemical alteration that can occur in response to cigarette smoking; however, little is known about the effect of SHS on human DNA methylation. In the present study, we evaluated the association between SHS exposure and DNA methylation in human monocytes, at a site (<span style=”font-style:italic;”>AHRR</span> cg05575921) known to have methylation inversely associated with current and former cigarette smoking compared to never smoking. Results from this study suggest high levels of recent SHS exposure inversely associate with DNA methylation of <span style=”font-style:italic;”>AHRR</span> cg05575921 in monocytes from nonsmokers, albeit with weaker effects than active cigarette smoking.</span>