Sepsis-associated encephalopathy (SAE) affects approximately one-third of septic patients, and there is a lack of effective therapeutics for SAE. Hydrogen gas is a new medical gas that exerts anti-inflammation, antioxidation, and anti-apoptotic effects and can effectively protect septic mice. Mitochondrial dysfunction, which can be improved by mitochondrial biogenesis, is a type of molecular pathology in sepsis. Peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α), which can be inhibited by SR-18292, is the key regulatory factor of mitochondrial biogenesis. Therefore, we investigated the effects of hydrogen gas on mitochondrial function and mitochondrial biogenesis in mice with SAE and the related regulatory mechanisms. Cecal ligation and puncture was used to induce sepsis in mice. The mice with hydrogen gas therapy were exposed to 2% H2 inhalation for 1 h beginning at both 1 and 6 h after operation, and mice were also injected with a PGC-1α inhibitor, SR-18292. We recorded the 7-day survival rates of the mice and detected their cognitive function using a Y-maze test. The Nissl bodies in the CA1 region of hippocampus were observed by Nissl staining, and the apoptotic cells were observed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay staining. The mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) level, and mitochondrial respiratory chain complexes I and II were analyzed using commercial kits. The mitochondrial morphology was observed by transmission electron microscopy. The expression levels of PGC-1α, nuclear respiratory factor 2 (NRF2), and mitochondrial transcription factor A (Tfam) were detected by Western blot analysis. The present study showed that hydrogen gas therapy increased the 7-day survival rate, improved cognitive function, increased the mitochondrial function (MMP, ATP level, complex I activity) and expression of mitochondrial biogenesis parameters (PGC-1α, NRF2, Tfam). However, the injection of SR-18292 (a PGC-1α inhibitor) decreased mitochondrial function, PGC-1α activation, and expression of NRF2 and Tfam. Therefore, these results indicate that hydrogen gas alleviates sepsis-induced brain injury in mice by improving mitochondrial biogenesis through the activation of PGC-1α.