Abstract: Maternal sleep deprivation (MSD) is a global public health problem that affects the physical and mental development of pregnant women and their newborns. The latest research suggests that sleep deprivation (SD) disrupts the gut microbiota, leading to neuroinflammation and psychological disturbances. However, it is unclear whether MSD affects the establishment of gut microbiota and neuroinflammation in the newborns. In the present study, MSD was performed on pregnant Sprague-Dawley rats in the third trimester of pregnancy (gestational days 15–21), after which intestinal contents and brain tissues were collected from offspring at different postnatal days (P1, P7, P14, and P56). Based on microbial profiling, microbial diversity and richness increased in pregnant rats subjected to MSD, as reflected by the significant increase in the phylum Firmicutes. In addition, microbial dysbiosis marked by abundant Firmicutes bacteria was observed in the MSD offspring. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) showed that the expression levels of proinflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were significantly higher in the MSD offspring at adulthood (P56) than in the control group. Through Spearman correlation analysis, IL-1β and TNF-α were also shown to be positively correlated with Ruminococcus_1 and Ruminococcaceae_UCG-005 at P56, which may determine the microbiota-host interactions in MSD-related neuroinflammation. Collectively, these results indicate that MSD changes maternal gut microbiota and affects the establishment of neonatal gut microbiota, leading to neuroinflammation in MSD offspring. Therefore, understanding the role of gut microbiota during physiological development may provide potential interventions for cognitive dysfunction in MSD-impacted offspring.