This research examined the association of cigarette smoking and altitude with the blood levels of 25-hydroxy vitamin D, testosterone and carotid artery thickness. Patients and methods: This comparative cross-sectional study involved 37 non-smokers and 24 smokers from a high-altitude area (≥2245 m above sea level) and 40 smokers and 40 non-smokers from a low-altitude area (39-283 m above sea level). The blood testosterone level was determined spectrophotometrically, and the 25-hydroxy vitamin D concentration was measured by ELISA. The IMT of the right and left carotid arteries was determined using ultrasound imaging. Results: Smoking notably elevated the thickness of the intima media of the right and left carotid arteries at both high and low altitudes (p ≤ 0.001). Smoking at high altitude was associated with a significant increase in the concentration of 25-hydroxy vitamin D and testosterone, while at low altitude it was associated with a significant decrease in both parameters (p ≤ 0.046). Conclusion:These contrasting results suggest that future studies should focus on finding out if other biochemical parameters show any significant differences in smokers or/and non-smokers when they are tested at elevated height and sea-level. This indicates that dose modifications of medicines (related to alterations in vitamin D and testosterone levels) should be kept in mind while treating smokers and non-smokers at elevated height above sea level.