Objective To investigate the protective effects and underlying mechanisms of berberine (BBR) on brain injury related to neonatal necrotizing enterocolitis (NEC). Methods An NEC mouse model was established and mice were assigned to control, model, low-dose BBR (2.5 mg/kg), and high-dose BBR (5 mg/kg) groups (n=24 per group). Body weight and survival were recorded. Hematoxylin-eosin staining was performed to assess pathological damage in intestinal and brain tissues; Nissl staining was performed to evaluate neuronal injury in the hippocampus; immunohistochemistry was conducted to detect microglial activation in the hippocampus. Lipopolysaccharide (LPS) levels in the intestine and brain were measured by ELISA. Western blot and quantitative PCR were used to quantify the protein and mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in brain tissue. Western blot was further used to examine the protein expression related to the TLR4/MyD88/NF-κB signaling pathway, NLRP3 inflammasome, and blood-brain barrier (BBB) tight junction proteins (ZO-1, Occludin, Claudin-5). Neurobehavioral functions were evaluated by the open field, novel object recognition, Y-maze, and Morris water maze tests. In vitro, BV2 microglial cells were divided into control, LPS, LPS+BBR, or LPS+TAK-242 (TLR4 inhibitor) groups (n=3 per group). Cell viability was assessed by CCK-8 assay and inflammatory signaling proteins were measured by Western blot. Results Compared with the model group, both BBR doses alleviated intestinal and brain pathology; the intestinal pathology score, escape latency on days 3 to 5, number of activated microglia (immunoreactive for ionized calcium-binding adaptor molecule 1), and TNF-α, IL-6, IL-1β mRNA/protein levels decreased significantly (P<0.05), while the spontaneous alternation rate increased (P<0.05). High-dose BBR reduced intestinal and brain LPS levels and downregulated the TLR4/MyD88/NF-κB pathway and NLRP3 inflammasome-related protein expression in the brain (P<0.05). During days 1 to 3 after NEC induction, body weight, central zone movement distance, central zone residence time, recognition index in the testing phase, platform crossings, percentage of time spent in the target quadrant, and expression of BBB tight junction proteins increased significantly in the high-dose group (P<0.05). In vitro, LPS+BBR and LPS+TAK-242 treatments reduced the protein expression related to the TLR4/MyD88/NF-κB pathway and NLRP3 inflammasome compared with LPS alone (P<0.05). Conclusions BBR can alleviate brain injury in NEC model mice and improve long-term neurological outcomes dose-dependently. These effects likely involve preservation of BBB integrity and suppression of microglial activation via inhibition of the TLR4/MyD88/NF-κB/NLRP3 signaling pathway.