The earliest effect of spaceflight is an alteration in vestibular function due to microgravity. Hypergravity exposure induced by centrifugation is also able to provoke motion sickness. The blood–brain barrier (BBB) is the crucial interface between the vascular system and the brain to ensure efficient neuronal activity. We developed experimental protocols of hypergravity on C57Bl/6JRJ mice to induce motion sickness and reveal its effects on the BBB. Mice were centrifuged at 2× g for 24 h. Fluorescent dextrans with different sizes (40, 70 and 150 kDa) and fluorescent antisense oligonucleotides (AS) were injected into mice retro-orbitally. The presence of fluorescent molecules was revealed by epifluorescence and confocal microscopies in brain slices. Gene expression was evaluated by RT-qPCR from brain extracts. Only the 70 kDa dextran and AS were detected in the parenchyma of several brain regions, suggesting an alteration in the BBB. Moreover, Ctnnd1, Gja4 and Actn1 were upregulated, whereas Jup, Tjp2, Gja1, Actn2, Actn4, Cdh2 and Ocln genes were downregulated, specifically suggesting a dysregulation in the tight junctions of endothelial cells forming the BBB. Our results confirm the alteration in the BBB after a short period of hypergravity exposure.