<i>Background/Aims:</i> Several lines of evidence indicate that sleep loss imposes significant consequences on the host defense system, including changes in cell number, activity and distribution. However, it is not clear whether cellular alterations after sleep deprivation are caused by redistribution to immune organs or by death of these cells or how the response to a nonspecific immune activator would be affected. Therefore, the aim of this study was to assess the leukocyte distribution after paradoxical sleep deprivation (PSD) in saline- and lipopolysaccharide-treated mice. <i>Methods:</i> Adult inbred mice were paradoxical sleep deprived (72 h), whereas the controls were kept in their home cages. After PSD, both groups received an injection of either saline or lipopolysaccharide (LPS; 1 or 5 µg/animal, intraperitoneally), 2 h prior to the collection of blood, spleen, lymph nodes and peritoneal wash. Isolated cells were then designated to differential leukocyte count (blood) and flow cytometry analysis of immune cell subsets (immune sites). <i>Results:</i> The data revealed that PSD caused a significant reduction of circulating lymphocytes and a general decrease in all cellular subsets of spleen, mainly T and B cells. However, no alteration in response of PSD was found on other immune sites, such as lymph nodes and peritoneum. Of note, immune cell distribution in response to in vivo LPS stimulation remained unchanged after PSD. <i>Conclusions:</i> Our study provided original evidence concerning the immune outcomes of PSD, indicating that cellular decrease caused by PSD is not restricted to circulation, but also to immune sites. Taken together, our results could help shed light on the physiological mechanisms of leukocyte trafficking.