<p>Diabetes-associated cognitive dysfunction (DACD) is a severe neurological complication of type 2 diabetes. Impaired autophagic flux is a critical contributor to the progression of DACD. This study aimed to clarify how tripartite motif–containing 25 (TRIM25) regulates autophagy in DACD. To mimic the hyperglycemic environment of diabetes, HT22 hippocampal neurons were exposed to high glucose (HG). Autophagic flux was monitored using an RFP–GFP–LC3 reporter. Transcriptional regulation was evaluated using chromatin immunoprecipitation and a dual-luciferase reporter assay. Protein stability and ubiquitination were examined using a cycloheximide chase assay and co-immunoprecipitation. Protein abundance was determined by western blot, whereas mRNA levels were quantified by reverse transcription quantitative polymerase chain reaction. In HT22 hippocampal neurons exposed to HG, TRIM25 expression was significantly upregulated, whereas sine oculis homeobox 2 (SIX2) and sprouty-related EVH1 domain–containing protein 2 (SPRED2) expression was significantly downregulated. TRIM25 directly interacted with SIX2 and promoted its ubiquitination and degradation. Moreover, TRIM25 inhibited neuronal autophagy under HG conditions by targeting SIX2. SIX2 transcriptionally activated SPRED2 expression and subsequently inhibited extracellular signal-regulated kinase (ERK) phosphorylation. HG-induced reductions in cell viability and autophagy were reversed by SIX2 overexpression but not when SPRED2 was simultaneously knocked down. TRIM25 reduces SPRED2 transcription by mediating the ubiquitination and degradation of SIX2, thereby exacerbating the suppression of neuronal autophagy under high-glucose conditions. This study identifies a novel TRIM25/SIX2/SPRED2/ERK axis through which hyperglycemia impairs neuronal autophagy.</p>