Water column hypoxia is a key process influencing methylmercury (MeHg) production and availability in waterbodies worldwide. During seasonal destratification, large, short-lived pulses of aqueous MeHg may be released into the subsequently mixed water column, but little is known about the fate of these pulses, particularly whether there are concomitant increases in MeHg uptake into aquatic food webs. We examined the magnitude and timing of MeHg uptake across several trophic guilds relative to the reservoir stratification status using biweekly mercury data from water, zooplankton, and fish (Bluegill, Lepomis macrochirus and Smallmouth Bass, Micropterus dolomieu). Zooplankton MeHg concentrations increased by up to 250% during destratification, concurrent with increases in aqueous MeHg concentrations. Zooplankton and filter-passing MeHg concentrations were positively correlated when the reservoir was mixed (R² = 0.95) and destratifying (R² = 0.57) but not while the reservoir was stratified (R² = 0.21). Mercury concentrations in adult bluegill and juveniles of both fish species increased 20–70% following destratification, with responses lagging 4–8 weeks behind those in water and zooplankton MeHg. Mercury concentrations in piscivorous adult bass varied little over the course of the study. Our findings demonstrate the responsiveness of reservoir food webs to pulses in MeHg availability, suggesting that these pulses could play an important role in biotic MeHg exposure within and downstream of reservoirs.