Extended space missions are known to induce stress and immune dysregulation. Hindlimb unloading is a ground-based model used to reproduce most spaceflight conditions. The aim of this study was to better characterize the consequences of prolonged exposure to hindlimb unloading on murine splenic lymphocyte sub-populations. To ensure that the observed changes were not due to tail restraint but to the antiorthostatic position, three groups of mice were used: control (C), orthostatic restrained (R) and hindlimb unloaded (HU). After 21 days of exposure, no difference in serum corticosterone levels nor in thymus and spleen weights were observed between HU mice and their counterparts, revealing a low state of stress. Interestingly, flow cytometric analyses showed that B cells were drastically reduced in HU mouse spleens by 59% and, while the T cells number did not change, the Th/Tc ratio was decreased. Finally, the use of a fluorescent dye monitoring lymphoproliferation demonstrated that lymphocyte response to mitogen was reduced in Th and Tc populations and to a greater extent in B cells. Thus, we showed for the first time that, even if restraint has its own effects on the animals and their splenic lymphocytes, the prolonged antiorthostatic position leads, despite the absence of stress, to an inversion of the B/T ratio in the spleen. Furthermore, the lymphoproliferative response was impaired with a strong impact on B cells. Altogether, these results suggest that B cells are more affected by hindlimb unloading than T cells which may explain the high susceptibility to pathogens, such as gram-negative bacteria, described in animal models and astronauts.