Lactobacillus reuteri FN041 is a secretory IgA-targeted Lactobacillus strain from human breast milk that has probiotic potential. The aim of this study was to test whether FN041 can alleviate dyslipidaemia and mucosal-barrier damage caused by a high-fat diet (HFD) and whether it can affect diurnal variation of the intestinal microbiota. C57BL/6 mice were fed either a normal chow diet or high-fat diet (HFD) for 7 weeks and were treated with either PBS as a control or L. reuteri FN041 for 4 weeks. Our results showed that FN041 treatment significantly attenuated HFD-induced weight gain (P < 0.01), accumulation of testicular fat, an increase in locomotor activity during the active phase (P < 0.01), triglyceridaemia, hypercholesterolaemia (P < 0.05), liver Fas overexpression, and Srebp1c mRNA expression inhibition. Moreover, FN041 treatment improved intestinal epithelial barrier function and induced a daily oscillation-dependent change in short-chain fatty acid production by the gut microbiota. A deeper understanding of the molecular pathways participating in intestinal barrier and microbiota modifications, and changes to lipid metabolism under the influence of FN041, will have important implications by potentially opening new horizons for the development of relevant foods to prevent metabolic disorders and unrelated intestinal diseases.