The effects of lake browning on trophic functioning of planktonic food webs are not fully understood. We studied the effects of browning on the response patterns of polyunsaturated fatty acids and n−3/n−6 ratio in seston and compared them between boreal and temperate lakes. We also compared the regional differences and the effects of lake browning on the reliance of zooplankton on heterotrophic microbial pathways and the mass fractions of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in zooplankton. Lake browning was associated with increasing phytoplankton biomass and concentrations of EPA and DHA in both temperate and boreal lakes, but the seston n−3/n−6 ratio was lower in temperate than boreal lakes, most likely due the differences in phytoplankton community composition. The browning-induced increase in phytoplankton biomass was associated with increased reliance of zooplankton on a heterotrophic microbial pathway for both cladocerans and copepods in boreal and temperate lakes. This increased reliance on the heterotrophic microbial diet was correlated with a decrease in the EPA and DHA mass fractions in temperate copepods and a decrease in the n−3/n−6 ratio in boreal cladocerans and copepods. Our results indicate that although phytoplankton responses to lake browning were similar across regions, this did not directly cascade to the next trophic level, where zooplankton responses were highly taxa- and region-specific. These results indicate that lake browning should be considered as an overarching moderator that is linked to, e.g., nutrient increases, which have more immediate consequences on trophic interactions at the phytoplankton–zooplankton interface.