Background: Atherosclerosis is a chronic inflammatory process characterized by a series of biological processes resulting in plaque formation in the vascular wall. The monocyte/macrophage might be an important key player in the initiation and progression of plaque formation. It has been shown that exposure to cigarette smoke (CS) increases the adhesion of monocytes to endothelial cells in vitro, a critical step in the initiation of atherosclerosis. Purpose: The purpose of this study was to investigate the molecular mechanisms underlying the effect of CS exposure on monocytic cells over time. Methods: Human Mono Mac 6 cells were treated with different doses (0.045 and 0.09 puff/ml) of CS that had been bubbled through phosphate buffered saline (PBS) or with control PBS for 30, 60, and 120 minutes. RNA from different cell samples was extracted for further transcriptomic analysis using the Human Genome U133 Plus 2.0 Affymetrix® gene expression platform. The statistical analysis, including a multiple linear model approach performed with the Limma package (Smyth GK,2005), identified 502 probesets (~428 genes) with a significant interaction (FDR ≤0.01) between the time and dose variables. Results: Genes involved in transcription and transcription regulation, cell cycle, endoplasmic reticulum- and oxidative-stress response were over-represented in the list of differentially regulated genes. Indeed, numerous genes involved in the Nrf2-mediated oxidative stress response and in the p53-signaling pathway were found to be coordinately activated in a dose- and time-dependent manner. In fact, the expression of the cycle-dependent kinase inhibitor p21 (CDKN1a) gene was found to be dose- and time-dependently increased, whereas the expression of some cyclins, targets of p21, was repressed by CS. Interestingly, CDKN1a is an important negative regulator of cell cycle and has been shown to be involved in monocytic cell differentiation to macrophages and to protect against atherosclerosis when knocked out in Apoe-/- mice. Conclusion: Overall, the results indicate that upon CS exposure, monocytic cells undergo stress responses and transcriptional gene expression regulation in a dose- and time-dependent manner, which may drive these cells to acquire new biological functionalities that may be involved in the development of atherosclerosis.