Aurantiochytrium limacinum 4W-1b (AL4W-1b) is a newly discovered microalgal strain with unique features. In the present study, we investigated the effects of ethanol extracts of AL4W-1b on lipopolysaccharide- (LPS-) induced inflammatory responses in RAW264 murine macrophage cells. Pretreatment of RAW264 cells with the AL4W-1b extract significantly reduced the production of LPS-induced nitric oxide (NO) and the expression of proinflammatory cytokine genes, including tumor necrosis factor α, interleukin- (IL-) 1β, and IL-6. Treatment with the AL4W-1b extract also decreased the production of IL-1β and IL-6. These results suggest that AL4W-1b might have anti-inflammatory effects in RAW264 cells. The NF-κB inhibitor, BAY 11-7082, synergistically prevented LPS-induced NO production after pretreatment with the AL4W-1b extract. Thus, the AL4W-1b extract may affect not only the NF-κB pathway but also other inflammatory pathways. To the best of our knowledge, this is the first study to report the anti-inflammatory effects of AL4W-1b extract and its mechanism of action in LPS-stimulated murine macrophage cells.