Purpose: Data on the relation between linoleic acid (LA) and alpha-linolenic acid (ALA) and type 2 diabetes mellitus (T2DM) risk are scarce and inconsistent. The aim of this study was to investigate the association of serum LA and ALA with fasting and 2 h post-load plasma glucose and glycated hemoglobin (HbA1c). Method: This study included 667 participants from third examination (2000) of the population-based Hoorn study in which individuals with glucose intolerance were overrepresented. Fatty acid profiles in serum total lipids were measured at baseline, in 2000. Diabetes risk markers were measured at baseline and follow-up in 2008. Linear regression models were used in cross-sectional and prospective analyses. Results: In cross-sectional analyses (n = 667), serum LA was inversely associated with plasma glucose, both in fasting conditions (B = −0.024 [−0.045, −0.002]) and 2 h after glucose tolerance test (B = −0.099 [−0.158, −0.039]), but not with HbA1c (B = 0.000 [−0.014, 0.013]), after adjustment for relevant factors. In prospective analyses (n = 257), serum LA was not associated with fasting (B = 0.003 [−0.019, 0.025]) or post-load glucose (B = −0.026 [−0.100, 0.049]). Furthermore, no significant associations were found between serum ALA and glucose metabolism in cross-sectional or prospective analyses. Conclusions: In this study, serum LA was inversely associated with fasting and post-load glucose in cross-sectional, but not in prospective analyses. Further studies are needed to elucidate the exact role of serum LA and ALA levels and dietary polyunsaturated fatty acids in glucose metabolism.