Polyunsaturated fatty acid (PUFA) consumption is recommended as part of a healthy diet, but evidence of the impact of individual species and biological concentrations on cognitive function is limited. We examined prospective associations of PUFA erythrocyte composition and dietary intake with measures of cognitive function among participants of the Boston Puerto Rican Health Study (aged 57 years). Erythrocyte and dietary PUFA composition were ascertained at baseline and associated with 2-year scores on the Mini-Mental State Exam (MMSE) (n = 1032) and cognitive domain patterns derived from a battery of tests (n = 865), as well as with incidence of cognitive impairment. Erythrocyte and dietary n-3 PUFA were not significantly associated with MMSE score. However, total erythrocyte and dietary n-3 very-long-chain fatty acids (VLCFA), and intake of individual species, were associated with better executive function (P-trend < 0.05, for all). There was evidence that greater erythrocyte n-6 eicosadienoic acid concentration was associated with lower MMSE and executive function scores (P-trend = 0.02). Only erythrocyte arachidonic acid (ARA) concentration predicted cognitive impairment (Odds Ratio = 1.26; P = 0.01). Among Puerto Rican adults, we found that n-3 VLCFA consumption may beneficially impact executive function. Further, these findings provide some evidence that n-6 metabolism favoring greater ARA tissue incorporation, but not necessarily dietary intake, could increase the risk of cognitive impairment.