Tuberculosis has severe impacts on both humans and animals. Understanding the genetic basis of survival of both Mycobacterium tuberculosis, the human-adapted species, and Mycobacterium bovis, the animal-adapted species, is crucial to deciphering the biology of both pathogens. There are several studies that identify the genes required for survival of M. tuberculosis in vivo using mouse models; however, there are currently no studies probing the genetic basis of survival of M. bovis in vivo. In this study, we utilize transposon insertion sequencing in M. bovis AF2122/97 to determine the genes required for survival in cattle. We identify genes encoding established mycobacterial virulence functions such as the ESX-1 secretion system, phthiocerol dimycocerosate (PDIM) synthesis, mycobactin synthesis, and cholesterol catabolism that are required in vivo. We show that, as in M. tuberculosis H37Rv, phoPR is required by M. bovis AF2122/97 in vivo despite the known defect in signaling through this system. Comparison to studies performed in species that are able to use carbohydrates as an energy source, such as M. bovis BCG and M. tuberculosis, suggests that there are differences in the requirement for genes involved in cholesterol import (mce4 operon) and oxidation (hsd). We report a good correlation with existing mycobacterial virulence functions but also find several novel virulence factors, including genes involved in protein mannosylation, aspartate metabolism, and glycerol-phosphate metabolism. These findings further extend our knowledge of the genetic basis of survival in vivo in bacteria that cause tuberculosis and provide insight for the development of novel diagnostics and therapeutics. IMPORTANCE This is the first report of the genetic requirements of an animal-adapted member of the Mycobacterium tuberculosis complex (MTBC) in a natural host. M. bovis has devastating impacts on cattle, and bovine tuberculosis is a considerable economic, animal welfare, and public health concern. The data highlight the importance of mycobacterial cholesterol catabolism and identify several new virulence factors. Additionally, the work informs the development of novel differential diagnostics and therapeutics for TB in both human and animal populations.