The gut microbiota critically confers various health benefits, whereas environmental chemicals can affect its constitution and functionality thereby increasing disease risk. In the present study, we aim to evaluate the toxic effects of a wildly-used herbicide 2,4-D (2,4-dichlorophenoxyacetic acid) on the gut microbiome and host using an occupationally relevant dose. A mouse model was used combined with metagenomic sequencing and metabolomic profiling to examine the alterations induced by subchronic low-dose 2,4-D exposure in fecal and plasma samples. The metagenomics results revealed a distinct gut microbial community with profound changes in diverse microbial pathways including urea degradation, amino acid and carbohydrate metabolism in 2,4-D-treated mice. Moreover, the metabolomics results revealed that the metabolic profiles in treatment group were differentiated from control group in both fecal and plasma samples. Toxic effects on the host of 2,4-D at an occupationally relevant dose were observed indicated by decreased acylcarnitine levels in plasma. These findings indicated that 2,4-D can cause toxicity and substantially impact the gut microbiome in mice at occupationally relevant doses, inferring that the relationship between environmental contaminants and microbiota is largely underestimated calling for more comprehensive consideration of the toxicity of occupational exposures.