Aim To study the health effects of road traffic noise on rats and the effects on their metabolite levels in vivo, and to investigate the possible mechanism. Methods Twelve Wistar rats (half male and half female) were randomly divided into noise group and control group according to sex. The noise group was exposed to road traffic noise at a mean of 75 dB from 23:00 daily to 7:00 the next day for 2.5 months. Blood pressure was measured, and feces were collected for LC-MS non-targeted metabolomics analysis. Results The systolic and diastolic blood pressure were higher in the noise group (P＜0.001). The mean systolic and diastolic blood pressure were (176.67±27.07) mmHg and (93.93±21.54) mmHg in noise exposed group, as well as (150.38±17.98) mmHg and (67.72±24.67) mmHg in control group. Metabolomic analysis identified 41 significant metabolites, including 2-hydroxyphenylacetic acid, N-formylmethionine, 3-hydroxyphenylalanine, etc., which mainly involved in the amino acid metabolic pathway. Conclusion Road traffic noise exposure was associated with a high blood pressure in rats. N-formylmethionine and 2-hydroxyphenylacetic may play roles in this association.