This study aimed to explore whether vitamin B complex (folic acid, B6 , and B12 ) could avert DNA methylation changes associated with inflammation induced by acute PM2.5 exposure. Sprague-Dawley rats were administered by gavage with different concentrations of vitamin B complex once a day for 28 days, and then by intratracheal instillation with saline or PM2.5 once every 2 days for three times. Vitamin B continued to be taken during the PM2.5 exposure. Rats were sacrificed 24 h after the last exposure. The results showed that vitamin B complex could block the pathological changes and injury in lungs induced by PM2.5 . Meanwhile, vitamin B complex could prevent the abnormal DNA methylation of IL-4 and IFN-γ to antagonize the imbalance of IL-4/IFN-γ associated with inflammation. It was further found that vitamin B complex could regulate DNA methyltransferases (DNMTs) and increase the S-adenosylmethionine (SAM)/S-Adenosyl-L-homocysteine (SAH) ratio to reverse the hypomethylation of genomic DNA and the abnormal DNA methylation of IL-4 and IFN-γ. In conclusion, vitamin B complex has a protective effect on acute lung injury by attenuating abnormal DNA methylation induced by PM2.5 in rats. This study may provide a new insight into the physiological function of vitamin B to prevent the health effects induced by PM2.5 .