Background and objective: While Chlamydia pneumoniae infection and hyperhomocysteinemia have been shown to contribute independently to the atherosclerotic risk, recent evidence has linked the association of C. pneumoniae positivity and hyperhomocysteinemia in patients with established atherosclerosis. The aim of this study was to investigate whether such a relationship can be replicated in India, where both infections and hyperhomocysteinemia are prevalent. Methods: Patients of acute ischemic stroke enrolled consecutively and prospectively in the Nizam’s Institute Stroke Registry, Hyderabad, India (NISHI) were subjected to thorough clinical and neuroimaging evaluation. Blood was drawn in fasting state for estimation of homocysteine level and the titers of C. pneumoniae antibodies (IgG and IgA) by microimmunofluorescence method. Results: Of the 200 stroke patients, 72 (36%) were tested positive for C. pneumoniae antibodies, and 128 (64%) tested negative. The percentage of subjects with hyperhomocysteinemia, smoking, hypercholesterolemia and C-reactive protein positivity was higher in C. pneumoniae positive group compared with C. pneumoniae negative group. Multiple logistic regression analysis showed that hyperhomocysteinemia was an independent variable in the C. pneumoniae positive group (Odds ratio 4.71 95% CI 2.2-9.8). Conclusion: This study has shown that C. pneumoniae seropositivity is linked with hyperhomocysteinemia in patients with ischemic stroke in a sample of South Indian population.