The gut–brain axis is currently being studied as a therapeutic strategy for neurological diseases, especially Alzheimer’s disease (AD). Obesity results in the gut microbiota dysbiosis, which includes butyrate-producing bacteria are reduced. Although sodium butyrate (NaB) has emerged as the potential therapeutic substance in AD, there is a lack of detailed results into what signaling pathways affect amyloidogenesis in AD induced by obesity. Thus, we investigated the regulatory role of NaB on amyloidogenesis in neuronal cells under high cholesterol. In our results, we verified that increased amyloid β peptide (Aβ) accumulation in the brain of obese mice and a reduction in butyrate-producing bacteria due to the gut microbiota dysbiosis induced by obesity. We showed that NaB decreased the expression levels of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and Aβ accumulation induced by high cholesterol in SK-N-MC cells. We demonstrated that NaB was absorbed in cells through sodium-coupled monocarboxylate transporter 1 (SMCT1) and then inhibited high cholesterol-induced Aβ accumulation. Subsequently, we also observed that reactive oxygen species (ROS) were overproduced because of increased NADPH oxidase 2 (NOX2) expression under high cholesterol. Meanwhile, NaB decreased NOX2 levels through a reduction of NF-κB activity, which ultimately inhibited Aβ accumulation caused by high cholesterol. We demonstrated that NaB increased the expression levels of p21 under high cholesterol, contributing to p21/NRF2 (Nuclear factor erythroid 2-related factor 2) colocalization, which leads to NRF2 stabilization. NRF2 stabilization causes NF-κB inactivation, followed by NOX2 suppression and superoxide dismutase 1 (SOD1) upregulation. Thus, NaB with SOD1 silencing under high cholesterol did not eliminate excessive ROS, and eventually resulted in Aβ accumulation. In conclusion, we demonstrated that NaB prevents excessive ROS through NOX2 suppression and SOD1 upregulation by p21/NRF2 pathway, which is critical for inhibiting BACE1-dependent amyloidogenesis in neuronal cells exposed to high cholesterol environment.