Although n-3 polyunsaturated fatty acids (n-3 PUFAs) have potential anti-insulin resistance activity, the mechanism remains largely unknown. In this study, increased glucose resistance, insulin sensitivity, and lower glycemia were observed upon streptozotocin (STZ) treatment in n-3 PUFA-enriched fat-1 mice compared to wild type (WT) mice. Endogenous n-3 PUFAs in fat-1 mice were found to impair hyperglycemia or high glucose level-induced nucleotide-binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) inflammasome activation and inhibit IL-1β secretion in adipose tissues. In addition, endogenous n-3 PUFAs also inhibited high glucose-induced caspase-1 activity and IL-1β secretion in pre-adipocyte-enriched stromal vascular fractions (SVF) isolated from adipose tissues. Furthermore, in 3T3-L1 pre-adipocytes, high levels of glucose induced thioredoxin interacting protein (TXNIP) expression and activated the NLRP3 inflammasome, which was counteracted by docosahexaenoic acid (DHA), the major n-3 PUFA in fat-1 mice, by downregulating TXNIP via the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Our results suggest that n-3 PUFA-mediated insulin sensitivity is at least partly associated with inflammasome inhibition in pre-adipocytes. Our findings highlight the potential clinical use of dietary n-3 PUFAs in the prevention or intervention of T2D and other NLRP3 inflammasome-driven inflammatory diseases.