Knowledge of the reproductive biology of wild sturgeon populations is critical to ensure the survival of this unique group of animals. We combined gill-netting surveys, nonlethal blood sampling, radiotelemetry, and egg collection to examine the reproductive biology of lake sturgeon (Acipenser fulvescens Rafinesque, 1817) at a suspected spawning ground below a dam on the Richelieu River, Quebec. Lake sturgeon were present at the beginning of sampling in early May, and spawning took place from 26 May to 5 June when water temperature averaged 13.4 ± 0.1 °C (range 11.5–15.5 °C). Daily spawning population estimates ranged from 285 to 1282 individuals and the sex ratio of spawners was estimated at 2.1 males per female. The presence of radio-tagged individuals on the spawning grounds peaked from 20 to 28 May, corresponding with known spawning bouts. Residence time of spawners on the spawning ground ranged from 1 to 27 days (median = 5 days) and there were no differences in residence time between sexes. Nonlethal blood sampling enabled the quantification of steroid levels to determine the spawning population sex ratio, and steroid levels were highest before spawning was known to occur and decreased concurrently with, and after, known spawning events.