Background: This study aimed at assessing the effect of a low-fat diet (LFD) in obese mice lacking toll–like receptors (Tlr) and understanding the expression and regulation of microRNAs during weight reduction. Methods: C57BL/6, Tlr5−/−, Tlr2−/− and Tlr4−/− mice were used in this study. A group of mice were fed with a high-fat diet (HFD) (58% kcal) for 12 weeks to induce obesity (diet-induced obesity, DIO). Another group that had been fed with HFD for eight weeks (obese mice) were switched to a low-fat diet (LFD) (10.5% kcal) for the next four weeks to reduce their body weight. The control mice were fed with a standard AIN-76A diet for the entire 12 weeks. The body weight of the mice was measured weekly. At the end of the experiment, epididymal fat weight and adipocyte size were measured. The differentially expressed miRNAs in the fat tissue was determined by next-generation sequencing with real-time quantitative reverse transcription polymerase chain reaction (RT–qPCR). Target prediction and functional annotation of miRNAs were performed using miRSystem database. Results: Switching to LFD significantly reduced the body weight and epididymal fat mass in the HFD-fed C57BL/6 and Tlr5−/− mice but not in Tlr2−/− and Tlr4−/− mice. Weight reduction significantly decreased the size of adipocytes in C57BL/6 but not in the Tlr knockout mice. In Tlr2−/− and Tlr4−/− mice, feeding with HFD and the subsequent weight reduction resulted in an aberrant miRNA expression in the epididymal fat tissue unlike in C57BL/6 and Tlr5−/−. However, target prediction and functional annotation by miRSystem database revealed that all the top 10 Kyoto Encyclopedia of Genes and Genomes (KEGG) database pathways of the dysregulated miRNAs during weight reduction in the C57BL/6 mice were also found in the regulated pathways of Tlr5−/−, Tlr2−/−, and Tlr4−/− strains. However, among these pathways, gene sets involved in arginine and proline metabolism and glutathione metabolism were mainly involved in the Tlr knockout mice but not in the C57BL/6 mice. Conclusions: In this study, we demonstrated that feeding of LFD leads to significant body weight reduction in C57BL/6 and Tlr5−/− mice, but not in Tlr2−/− and Tlr4−/− mice. Significant reduction in the size of adipocytes of epididymal fat was only found in C57BL/6, but not in Tlr5−/−, Tlr2−/−, and Tlr4−/− mice. The dysregulated miRNAs in Tlr2−/− and Tlr4−/− mice were different from those in C57BL/6 and Tlr5−/− strains. Among those miRNA-regulated pathways, arginine and proline metabolism as well as glutathione metabolism may have important roles in the Tlr knockout mice rather than in C57BL/6 mice.