Abstract Background The aim of the study was to compare acute endothelial dysfunction caused by smoking one cigarette between smokers and non-smokers. Methods Thirty volunteers, 15 smokers and 15 non-smokers, were asked to smoke the same type of cigarette and endothelial function was assessed by FMD (flow mediated dilatation) at fast, 30, 60 and 90 min post-smoking. Results Overall response of FMD after smoking was significantly different between smokers and non-smokers ( p = 0.011). Endothelial dysfunction after smoking remained significant for 1-h in smokers ( p = 0.002), but only for 30 min in non-smokers ( p = < 0.001). FMD 1-h after smoking was significantly higher in non-smokers than in smokers ( p = 0.002). Conclusions Smokers seem to have sustained and more intensive unfavourable effects in endothelial function than non-smokers after smoking one cigarette. It is possible that long-term smoking diminishes the capacity of endothelium to counterbalance external factors that cause endothelial dysfunction.