Abstract. Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) are associated with high morbidity and mortality in patients, however, the precise pathogenesis of ALI/ARDS remains unknown. Lipopolysaccharide (LPS) exhibits a number of critical functions and may be associated with the DNA methylation of genes in the lungs. In the present study a genome-wide analysis of DNA methylation was performed in rat lungs with LPS-induced ALI/ARDS. Normal and LPS-induced lung tissues with ALI were analyzed using methylated DNA immunoprecipitation and a rat DNA methylation promoter plus CpG island microarray and the candidate genes were validated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Aberrant DNA methylation of the promoter regions of 1,721 genes and the CpG islands of 990 genes was identified when normal lung tissues and lung tissues with LPS-induced ALI/ARDS were compared. These genes were commonly located on chromosomes 1, 3, 5, 7 and 10 (P<0.01). Methylation level and CpG density were compared and it was found that genes associated with high CpG density promoters had a high ratio of methylation. Furthermore, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In addition, three genes (Mapk3, Pak1 and Rac2) were validated in the control and lung tissues with ALI by RT-PCR. The results indicate that aberrant DNA methylation of lung tissues may be involved in the pathophysiology of LPS-induced ALI/ARDS. Future studies are required to evaluate the therapeutic and prognostic value of the current novel observations in ALI/ARDS.