The mammal fauna of the Willwood Formation, central Bighorn Basin, Wyoming, is ideal for paleoecological analysis because it is extensive, well studied, and continuously distributed over sediments representing the first 3 Myr of the early Eocene. The geology of the Bighorn Basin is also well known, providing a precise temporal framework and climatic context for the Willwood mammals. Previous analysis identified three “biohorizons,” based on simple counts of the first and last appearances of species. This study uses species diversity and appearance rates calculated from more extensive collections to approximate the ecological dynamic of the ancient fauna and assess whether the biohorizons were significant turnover events related to recently described climatic variation. Diversity and appearance data collected for this project are extensively corrected for uneven sampling, which varies by two orders of magnitude. Observed, standardized appearance and diversity estimates are subsequently compared with predicted background frequencies to identify significant variation. Important coincident shifts in the biotic parameters demonstrate that ecological change was concentrated in two discrete intervals ≤300 Kyr each that correspond with two of the original biohorizons. The intervals coincide with the onset and reversal of an episode of climate cooling identified directly from Bighorn Basin floras and sediments. Ecological changes inferred from the diversity and turnover patterns at and following the two biohorizons suggest short- and long-term faunal response to shifts in mean annual temperature on the order of 5–8°C.