Emerging studies indicate that cardiovascular function shows diurnal variations and the levels of several genes and metabolites oscillate in a circadian manner. However, little is known about the effect of circadian sensitivity on smoking induced alterations in cardiovascular function and pathology, especially in experimental animals. We exposed 8 week old C57BL/6J mice to mainstream cigarette smoke MCS (Kentucky 3R4F cigarettes) during the light (inactive cycle) or dark (active cycle) light conditions (12 weeks, 7 day/week, 6 hours/day). Circadian rhythm sensitive genes Bmal-1 and Cry-1 were 5.1-5.6-fold higher in the lungs of air-exposed mice in their active cycle than in the inactive cycle (P<0.05). In the active phase, MCS exposure increased the expression of Per1, Per2 and Per3 genes by 1.7-3.4 fold (P<0.05) as compared with MCS exposure in the inactive phase. MCS-exposure did not affect body weight in light or dark cycle. Levels of total nicotine equivalent (nicotine and its metabolites) in the plasma were increased by 1.5-fold (P<0.05) in mice exposed to MCS in the active phase than inactive phase. Analyses of complete blood count showed that MCS-exposure significantly decreased neutrophils by 40% in the active phase and 28% in the inactive phase. White blood cells and lymphocytes were decreased by 35-37% (P<0.05) only in the mice exposed to MCS in the active phase (P<0.05). Flow cytometric analyses showed that MCS-exposure decreased the levels of NK cells, granulocytes, B-cells and monocytes (by 57-68%; P<0.05) only in the mice exposed to MCS in the active phase. Circulating endothelial progenitor cells (EPCs) were depleted by 48% (P<0.05) in mice exposed to MCS in the active phase. On the contrary, plasma cholesterol levels and aortic lesion formation were increased by 21% (P<0.05) and 60% (P<0.05) in apoE-KO mice exposed to MCS in the inactive phase than the active phase. Collectively, our data suggest that, circadian rhythm is a critical regulator of MCS-induced cardiovascular functions and pathology.