Smoking is recognized as a major risk factor for developing cardiovascular diseases. Smoking causes a reduction in nitric oxide (NO) together with an unbalance in the autonomic nervous system (ANS). The changes lead to an attenuated ability to regulate vascular tonus in order to meet various metabolic demands. Research indicates that ischemic preconditioning (IPC) has positive effects on both NO and the ANS. IPC consists of multiple cyclicals of short periods of restriction in blood supply to the implicated tissue and subsequent reperfusion. The effect of IPC on microvascular function and the contribution of the ANS in smokers and non-smokers has not yet been investigated. Hence, the aim of this study was to determine the effect of IPC on microvascular function in young healthy smokers and non-smokers and elucidate the contribution of the ANS. Eighteen young healthy smoking (n = 7) and non-smoking (n = 11) males went through controlled counter balanced crossover design over two sessions. The participants received either IPC (250 mmHg) or sham conditioning (20 mmHg) at the first session and vice versa at the second session. Each session consisted of three measurement blocks (Baseline, Post, and Post 1-hour). At each measurement block, microvascular function and the contribution of the ANS was measured. Microvascular function was assessed as the contraction-induced hyperemic responses to single brief contractions of the dorsiflexor muscles using near-infrared spectroscopy (HbO2). The contribution of the ANS was assessed via heart rate variability (high frequency and low frequency domains) using electrocardiographic recordings. IPC or sham conditioning were performed between Baseline and Post. HbO2 time-to-peak and HbO2 relative peak were increased for both groups and conditionings at Post and Post 1-hour compared to Baseline (all p’s < 0.001 for both time-to-peak and relative peak). No change for the high frequency domain was found for both groups and conditionings (all p’s ≥ 0.065). A significant increase in the low frequency domain was found for both groups and conditionings at Post and Post 1-hour (all p’s ≤ 0.027). Microvascular function was not affected acutely by IPC. Neither were there any differences found between smokers and non-smokers. IPC did not alter the balance of the ANS. Smokers and non-smokers did not exhibit different autonomic tone at any time. Inactivity seems to play a pivotal role in explaining the changes found in this study. In the future the intensity and duration of the physical demand should be considered when studying differences between smokers and non-smokers. Furthermore, the dosage of IPC seems to be important for achieving microvascular adaptations.