During a 17-yr telemetry study, we examined the diet and ambush behavior of a population of Crotalus horridus in southeastern Virginia. Forty dietary items were identified from 37 fecal samples. We documented 722 instances of snakes in an ambush posture, 61% of which were in a vertical-tree posture, as if hunting arboreal prey at the base of a tree. The most common prey items were Eastern Gray Squirrels (Sciurus carolinensis), which accounted for 45% of all dietary items and represented an estimated 78% of total biomass consumed by C. horridus. Prey was not consumed in proportion to availability, based on small mammal surveys. Our analysis provides indirect evidence that the vertical-tree foraging behavior is adopted to target arboreal Eastern Gray Squirrels. Further, we provide support for the hypothesis that C. horridus alters ambush behavior to forage selectively for specific prey types.