The high level of docosahexaenoic acid (DHA) in phospholipids in the brain has spurred significant interest in its role in brain health and diseases. Recent studies provided evidence for DHA to play a role in inhibiting inflammatory responses in microglial cells, although the mechanism(s) remains elusive. In this study, a global proteomic approach was used to examine effects of DHA on microglial cells stimulated with lipopolysaccharides (LPS). Deep proteome coverage was achieved using the parallel accumulation serial fragmentation (PASEF) method in a hybrid trapped ion mobility spectrometry (TIMS) – quadrupole time-of-flight mass spectrometer (TIMS–ToF). Using label-free quantitative proteomics, a total of 2858 protein groups (with more than one unique peptide) were confidently identified and quantified in BV-2 microglial cells. Treating the cells with LPS and/or DHA altered cell morphology and expression of 43 proteins with a differential abundance (greater than 2.0-fold change). Bioinformatic analyses further indicated that these differentially abundant proteins were involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, fatty acid metabolism, mitochondrial activity, response to bacterium, cytoskeleton, DNA binding, and ribosome biogenesis. Quantitative analyses of cell viability, tumor necrosis factor alpha, phospho-NF-κB p65, inducible nitric oxide synthase and prostaglandin E2 expression levels were consistent with the biological outcomes of the altered protein concentrations. Together, these data indicate for the first time multiple mechanisms of DHA-mediated protective effects in LPS-stimulated BV-2 microglial cell at the global proteome level. This information may shed light on a new basis for therapeutic strategies of DHA against inflammation.