Preterm labor is associated with inflammation and infection. The mechanisms underlying the role of omega-3 fatty acid in inflammasome activation and prevention of preterm labor remain unknown. We hypothesized that omega-3 fatty acid can reduce the rate of preterm birth induced by infection and trophoblast inflammation. In this study, we found that inflammasome-related molecules and IL-1beta in trophoblasts were activated by TNF-alpha derived from lipopolysaccharide-stimulated THP-1 cell-conditioned medium and recombinant TNF-alpha protein. The results demonstrated that stimulation with TNF-alpha caused lysosomal rupture in trophoblasts, which accelerated cathepsin S diffusion from lysosomes to the cytosol and activated NLRP1 and AIM2 inflammasomes, thereby increasing IL-1beta secretion. Moreover, in response to LPS challenge, TNF-alpha increased trophoblast cell death and decreased cell viability through inflammasome and cathepsin S activation. Stearidonic acid (SDA; 18:4n-3) and docosahexaenoic acid (DHA; 22:6n-3) inhibited inflammasome-related molecule synthesis and cathepsin S and caspase-1 activation, which further reduced the preterm delivery rate of pregnant mice induced by lipopolysaccharide (92.9% vs 69.7% [DHA]; 92.9% vs 53.5% [SDA]). Higher expression of TNF-alpha, IL-1beta, prostaglandin E2, and cathepsin S, but lower resolvin D1 expression, was observed in preterm pregnant mice than in controls. Similarly, resolvin D1 was highly expressed in women with term delivery compared with women with preterm delivery. Thus, SDA and DHA may attenuate macrophage-derived TNF-alpha inducing cathepsin S and inflammasome activation, IL-1beta secretion and placental trophoblast cell death. These functions are implicated in the preventive effects of SDA and DHA on preterm labor.