<b><i>Background:</i></b> Globally, 1 in 11 adults has diabetes mellitus, and most of these cases are type 2 diabetes (T2D). The risk of T2D is influenced by many factors, including diet. The synthesis of long-chain n-6 polyunsaturated fatty acids (LC n-6 PUFA) has been posited as a risk factor for T2D; however, its causal role is uncertain. <b><i>Aim:</i></b> To test the causal effect of LC n-6 PUFA synthesis on insulin resistance and transgenerational T2D risk in a large cohort of men and women. <b><i>Methods:</i></b> Two-sample mendelian randomization (MR) was conducted to evaluate the effect of low or high levels of LC n-6 PUFA synthesis on glycemia and development of T2D in the UK Biobank (<i>n</i> = 463,010) and Meta-Analysis of Glucose- and Insulin-Related Traits Consortium (MAGIC; <i>n</i> = 5,130) cohorts. The increased likelihood of a predisposition to low or high LC n-6 PUFA synthesis and the risk of T2D was also investigated using the participants’ siblings and parents. In MR-Base, 4 genetic variants associated with LC n-6 PUFA synthesis were found (<i>p</i> &#x3c; 10^–8). After pruning, 1 variant (rs174547) on the FADS1 gene was retained. <b><i>Results:</i></b> Lower LC n-6 PUFA synthesis and abundance (per % unit decrease) are associated with small reductions in the insulin disposition index (–0.038 ± 0.012 mM^–1; <i>p</i> = 0.002) within MAGIC. In the UK Biobank, we report negligible effects of low n-6 PUFA synthesis on the odds of T2D (OR &#x3c;1%; <i>p</i> &#x3c; 0.05). Additionally, reduced LC n-6 PUFA synthesis does not appear to be a contributor to familial T2D risk. No significant association was observed between LC n-6 PUFA synthesis and BMI. <b><i>Conclusion:</i></b> In a primarily white European population, LC n-6 PUFA synthesis is not a major contributor to T2D risk.