Consumers feeding at the aquatic-terrestrial ecosystem interface may obtain a mixture of aquatic and terrestrial diet resources that vary in nutritional composition. However, in lake riparian spiders, the relative significance of aquatic versus terrestrial diet sources remains to be explored. We investigated the trophic transfer of lipids and polyunsaturated fatty acids (PUFA) from emergent aquatic and terrestrial insects to spiders at varying distances from the shoreline of a subalpine lake in Austria, using differences in fatty acid profiles and compound-specific stable carbon (delta C-13) and hydrogen (delta H-2) isotopes. The omega-3 PUFA content of emergent aquatic insects was higher than that of terrestrial insects. Emergent aquatic insects contained on average 6.6 times more eicosapentaenoic acid (EPA) and 1.2 times more alpha-linolenic acid (ALA) than terrestrial insects, whereas terrestrial insects contained on average 2.6 times more linoleic acid (LIN) than emergent aquatic insects. Spiders sampled directly on the lake and in upland habitats had similar EPA contents, but this EPA was derived from different diet sources, depending on the habitat. The delta C-13(EPA) and delta H-2(EPA) values of 'lake spiders' revealed an aquatic diet pathway (i.e. EPA of aquatic origin). In contrast, EPA of spiders collected in terrestrial habitats was depleted in both C-13 and H-2 compared to any potential food sources, and their ALA isotopic values, suggesting that EPA was partly bioconverted from its dietary precursor ALA (i.e. internal pathway). The delta H-2 values of fatty acids clearly indicated that diet sources differed depending on the spider's habitat, which was less evident from the delta C-13 values of the fatty acids. Our data highlight that spiders can use two distinct pathways (trophic versus metabolic) to satisfy their physiological EPA demand, depending on habitat use and dietary availability.