We use a combination of spectral sensitivity analyses, laboratory behavioral observations and field distributions of a vertically migrating invertebrate, Hemimysis anomala (a recent invasive species to the Laurentian Great Lakes of North America), to determine if light preference and timing of emergence has an ontogenetic component. Juvenile Hemimysis (< 4.5 mm) preferred light levels between 10−3.4 and 10−2.4 mylux— a Hemimysis-specific unit of brightness derived from visual pigment analyses (wavelength of maximum absorbance = 500 nm; 1 mylux ~ 159 lx). These preferred light levels are equivalent to those present during nautical twilight on the Earth's surface and were several orders of magnitude brighter than those most preferred by adults (> 4.5 mm) in the laboratory (10−6.4 to 10−7.4 mylux). Both size classes completely avoided light levels of 10−0.4 mylux and greater, which are representative of daytime light levels at the Earth's surface. Net hauls taken at ~ 20-min intervals from sunset to the end of nautical twilight on two sampling occasions on Seneca Lake, New York (sampling depth = 2 m) revealed that juveniles emerged into the water column during civil twilight. Adult Hemimysis emerged later during nautical twilight when juveniles had already reached their maximum abundance in the water column. Laboratory-derived light preferences successfully predicted the timing of emergence and time of maximal abundance of both size classes on both sampling occasions. This study is one of the first to demonstrate that Hemimysis diel vertical migration has an ontogenetic component and to report the specific light levels likely to initiate and limit vertical movements.