Metabolic syndrome (MS) is a collection of risk factors of serious metabolic diseases. L-Carnitine is an essential nutrient for human health, and the precursor of trimethylamine N-oxide (TMAO). Previous studies have shown that the effect of L-carnitine on MS is controversial, and no studies have considered the role of gut microbiota in the regulation of MS by L-carnitine. In the present study, we established a high-fat diet (HFD)-induced obese mice model and systematically explored the effect of a broad range of dietary L-carnitine concentrations (0.2% to 4%) on the major components of MS. The results show that L-carnitine (0.5%-4%) reduced HFD-caused body-weight gain, visceral adipose tissue, glucose intolerance, hyperglycemia, HOMA-IR index, hyperlipemia, hypertension, and hyperuricemia. The elevation in the concentrations of IL-6, IL-1β, and TNF-α and decline in IL-10 in both serum and adipose tissue were also attenuated by L-carnitine. Furthermore, dietary L-carnitine increased the serum levels of TMAO produced by gut microbes. High-dose L-carnitine (2% and 4%), but not low-dose L-carnitine (0.2%-1%), notably modulated the composition of gut microbiota and partially attenuated HFD-induced gut microbiota dysbiosis. These results suggest that the ameliorative effect of L-carnitine on MS was independent of TMAO production and only partially related to the regulation of gut microbiota. This study provides crucial evidence for the utilization of L-carnitine as a safe and effective supplement for MS.