Dietary ratios of n-3/n-6 polyunsaturated fatty acids (PUFAs) have been implicated in controlling markers of metabolic disorders, including obesity, insulin resistance (IR), inflammation, and lipid profiles, which are also presumed to be partly related to type 2 diabetes mellitus (T2DM). However, molecular mechanisms of the different PUFAs related to metabolic disorders have not been systematically addressed. The present study aimed to investigate the impact of dietary n-3/n-6 PUFA ratios on obesity and IR and, further, to determine the underlying mechanisms. For 16 weeks, 32 SD male rats, randomly divided into four groups (n = 8 per group), received one of the following diets: normal chow, high saturated fatty acid (SFA), high n-3/n-6 PUFA ratio (1∶1, PUFA1:1), or low n-3/n-6 PUFA ratio (1∶4, PUFA1:4). Following the experimental diet period, metabolic parameters related to obesity and IR were measured. Compared to SFA diet-fed rats, PUFA1:1 diet-fed rats exhibited decreased body and visceral fat weight, lowered blood lipids, and improved glucose tolerance and insulin sensitivity. Interestingly, these changes were accompanied with decreased expression levels of circulating pro-inflammatory cytokines, including tumor necrosis factor α, interleukin-6, and C-reactive protein. Moreover, the TLR4 protein and mRNA levels were markedly down-regulated by PUFA1:1 compared with SFA; however, PUFA1:4 diet-fed rats failed to exhibit these changes. Cumulatively, our data highlight a role for a PUFA1:1 diet in the prevention of obesity and related metabolic disorders by suppressing the activation of TLR4, a critical modulator of pro-inflammatory cytokines.