Ammodytes personatus, known as the Pacific sand lance, thrives in cold areas of the North Pacific. In this study, the total lipid was extracted from A. personatus eggs and the fatty acid composition was determined using gas chromatography (GC)–flame ionization detection (FID). The results showed that the extracted lipid contained high content of polyunsaturated fatty acids (PUFAs). The immunomodulatory activities of the A. personatus lipid were investigated using rodent macrophages. First, immune enhancement was analyzed, and the A. personatus lipid significantly and dose-dependently increased the NO production in RAW264.7 cells, and this lipid also regulated the transcription of immune-associated genes in RAW264.7 cells by activating the NF-κB and MAPK pathways. Additionally, flow cytometry revealed that this lipid stimulated phagocytosis. Conversely, the anti-inflammatory activity of the A. personatus lipid was also analyzed and the results showed significantly decreased NO production and gene expression in a dose-dependent manner in LPS-stimulated RAW264.7 cells. In addition, the A. personatus lipid suppressed the LPS-induced phosphorylation of proteins related to the NF-κB and MAPK pathways in LPS-stimulated RAW264.7 cells. Further, flow cytometry demonstrated the lipid-regulated anti-inflammatory activity via inhibition of CD86 expression. The results indicate that A. personatus egg lipid is a potential source of immunomodulation.