Background-Previous studies demonstrated an association between antibodies to mycobacterial heat-shock protein 65 (mHSP65) and carotid artery thickening. We examined whether mHSP65 antibodies are associated with levels of coronary calcification that appear to reflect preclinical coronary artery disease (CAD). Methods and Results-Serum specimens from 201 healthy asymptomatic subjects (52% male; mean age, 56.6 years) undergoing electron-beam computed tomographic imaging were used to measure levels of mHSP65 and human HSP60 antibodies and antibodies to several infectious pathogens. We found that 84% of the study subjects had anti-mHSP65 IgG antibodies. Mean titers of mHSP65 antibodies were higher (1:394 versus 1:267, Pϭ0.012) in individuals with than in those without elevated levels of coronary calcium (calcium score Ն150). Increasing titers of mHSP65 antibodies were significantly associated, in a dose-response manner, with elevated levels of coronary calcification. Individuals with the highest titers of mHSP65 antibodies (Ն1:800) had an adjusted odds ratio (OR) of 14.3 for having elevated coronary calcium (Pϭ0.004). Association of mHSP65 antibodies with elevated coronary calcification levels was independent of CAD risk factors after multivariate adjustment (Pϭ0.037). Interestingly, mHSP65 antibody titers were correlated with Helicobacter pylori infection (Pϭ0.004), which maintained significance after adjustment for CAD risk factors and seropositivities to other pathogens (adjusted OR, 3.1; 95% CI, 1.4 to 6.6). No association was found between antibodies to human HSP60 and levels of coronary calcification. Conclusions-Antibodies to mHSP65 are associated with elevated levels of coronary calcification and correlated with H pylori infection, suggesting that pathogen-triggered autoimmunity plays a role in early atherosclerosis.