Increases in terrestrially-derived dissolved organic matter (DOM) have led to the browning of inland waters across regions of northeastern North America and Europe. Short-term experimental and comparative studies highlight the important ecological consequences of browning. These range from transparency-induced increases in thermal stratification and oxygen (O2) depletion to changes in pelagic food web structure and alteration of the important role of inland waters in the global carbon cycle. However, multi-decadal studies that document the net ecological consequences of long-term browning are lacking. Here we show that browning over a 27 year period in two lakes of differing transparency resulted in fundamental changes in vertical habitat gradients and food web structure, and that these responses were stronger in the more transparent lake. Surface water temperatures increased by 2-3 °C in both lakes in the absence of any changes in air temperature. Water transparency to ultraviolet (UV) radiation showed a fivefold decrease in the more transparent lake. The primary zooplankton grazers decreased, and in the more transparent lake were largely replaced by a two trophic level zooplankton community. These findings provide new insights into the net effects of the complex and contrasting mechanisms that underlie the ecosystem consequences of browning.