Background: The effect of traffic related emissions on Heart Rate Variability (HRV) has been previously demonstrated. However, the results of different studies on the relationship between ambient pollutants and specifically carbon monoxide (CO) on HRV are inconclusive and appear to reflect personal differences. The differences in methodology including breathing style and participants under study can possibly attribute to this variability. The aim of the present study is to assess the effect of the breathing route (Nasal/Oral) on the relationship between CO concentration and HRV. Methodology: Forty healthy participants (25 females, 15 males) between the ages of 15 - 50 years were included in the study. The participants strolled in a central bus station, staying for 10 minutes in three designated locations alternating nasal and oral breathing. CO concentration and HRV were continuously monitored. Frequency (LnLF, LnHF, LnLF/HF) and time domain (LnSDNN, LnRMSSD) HRV indices were computed. Analysis: MANCOVA, with HRV indices being the dependent variables and CO, gender and age being the independent variables was employed. Results: Significant interaction effects between breathing route and CO, and breathing route and gender on HRV (LnLF/HF) were found (p = 0.04 and 0.01 respectively). Both CO and age were found to affect LnSDNN and LnRMSSD. Conclusion: Breathing route emerges as a possible modifier of the relationship between air pollution and HRV and thus contributes to interpersonal differences obtained in studies investigating the effect of environmental pollution and HRV.