Intro: Prevalence of obesity is expanding world-wide. Obesity is a severe risk factor for fatty liver and brain diseases, and problems in mental health. The role of gut microbiota (GM) in health maintenance is widely accepted; thus, the modification of GM owns huge potential for treating many diseases. Feacaelibacterium prausnitzii is one of the gut microbes associating with low hepatic fat (Munukka et al. 2014), and suggested to act as a possible psychobiont, a tool to modify and support mental health (Borkent et al. 2022). Because F. prausnitzii is extremely anaerobic, the gut level of it should be targeted via dietary means. Aims/objectives: We previously showed that a prebiotic XOS enhanced growth of F. Prausnitzii and ameliorated hepatic steatosis in rats (Lensu et al. 2020). Here we show the effects of high-fat diet and/or XOS supplementation on behavior and cognition, before and during the dietary intervention in adult male Wistar rats. Method: The ethical permission for the study was achieved from the National Animal Experiment Board of Southern Finland. To enhance the level of F. Prausnitzii, the diet of the rats (n=10 per group) was supplemented or not with prebiotic dose of XOS (0.12%, Shandong Longlive Biotechnology, CAS 87099-0), and the rats were having a simultaneous high-fat diet (60% energy from fat) to induce obesity or control diet (10% energy from fat, ‘low-fat’), the details of the experiment can be found in Lensu et al. 2020. Behavior and cognition were studied with openfield, context-object recognition, and sucrose preference tasks. Untargeted metabolites from half of the brain tissue were measured by liquid chromatography – high resolution mass-spectrometry (LC-MS/MS) and curated using MS-DIAL and R. Data were tested with Generalized linear and mixed models (effect of high-fat diet and/or XOS, measurement time: pre-post) and between groups comparisons were done using non-parametric Kruskall-Wallis test. Results and conclusions: The activity of rats in the openfield-arena diminished during the 12-week intervention, independent of the group (F [1,34] = 44.2, p < 0.001), and low-fat diet enhanced anxiety-related behavior (F [1,35.8] = 5.4, p < 0.05). Low-fat diet attenuated the performance in the context-object recognition task in comparison to high-fat diet (F [1,36] = 14.45, p<0.001), explained by increased preference of the familiar object in the low-fat group during the post-measurement. In the end of the intervention, rats having high-fat diet preferred sucrose more than those having low-fat diet (F([1,36] = 6.59, p = 0.015). For the brain metabolites, the high-fat diet group showed separation from the other groups in principal component analysis and t-stochastic neighbor embedding. Using the pathway analysis in Metaboanalyst, eight metabolic pathways were found to be significantly different (q-value < 0.05 and pathway impact > 0.1) between high- and low-fat groups, including purine, pyridimine, histidine and tryptophan metabolism. In conclusion, our 12-week intervention caused only minor effects on behavior and brain metabolites, and they were mainly affected by high-fat diet. Thus, our research suggests that XOS is not highly psychoactive prebiotic although it beneficially affects the GM and host’s physiology.