Trimethylamine N-oxide (TMAO) is a risk factor of various chronic diseases, which was produced by metabolism from precursors to trimethylamine (TMA) in gut and the oxidation from TMA in liver. The TMA generation was influenced by diet, mainly due to the rich TMAO precursors in diet. However, it was still unclear that the effects of different proportion and source of macronutrients in different dietary pattern on the production process of TMAO. Here, the generation of TMA from precursors and TMAO from TMA was determined after single oral choline chloride and intraperitoneal injection TMA, respectively, in mice fed with carbohydrates, proteins and fats in different proportion and sources. The results suggested that the generation of TMAO was increased by low non-meat protein and high fat via enhancing the production of TMAO from TMA, and decreased by plant protein and refined sugar via reducing TMA production from precursors in gut and TMAO transformation from TMA in liver. The high fat and high sugar diets accelerating the development of atherosclerosis did not increase the production of TMAO, the risk factor for atherosclerosis, which indicated that the dietary compositions rather than the elevated TMAO level might be a more key risk factor for atherosclerosis.