Bacteroides fragilis comprises 1% 5% of the gut microbiota in healthy humans but can expand to >50% of the population in ulcerative colitis (UC) patients experiencing inflammation.The mechanisms underlying such microbial blooms are poorly understood, but the gut of UC patients has physicochemical features that differfrom healthy patients and likely impact microbial physiology. For example, levels of the secondary bile acid deoxycholate (DC) are highly reduced in the ileoanal J-pouch of UC colectomy patients. We isolated a B. fragilis strain from a UC patient with pouch inflammation(i.e., pouchitis) and developed it as a genetic model system to identify genes and pathways that are regulated by DC and that impact B. fragilis fitnessin DC and crude bile. Treatment of B. fragilis with a physiologically relevant concentration of DC reduced cell growth and remodeled transcription of one-quarter of the genome. DC strongly induced expression of chaperones and select transcriptional regulators and effluxsystems, and down-regulated protein synthesis genes. Using a barcoded collection of ∼50,000 unique insertional mutants, we further definedB. fragilis genes that contribute to fitnessin media containing DC or crude bile. Genes impacting cell envelope functions including cardiolipin synthesis, cell surface glycosylation, and systems implicated in sodium-dependent bioenergetics were major bile acid fitnessfactors. As expected, there was limited overlap between transcriptionally regulated genes and genes that impacted fitnessin bile when disrupted. Our study provides a genome-scale view of a B. fragilis bile response and genetic determinants of its fitnessin DC and crude bile. IMPORTANCE The Gram-negative bacterium Bacteroides fragilis is a common member of the human gut microbiota that colonizes multiple host niches and can influencehuman physiology through a variety of mechanisms. Identificationof genes that enable B. fragilis to grow across a range of host environments has been impeded in part by the relatively limited genetic tractability of this species. We have developed a high-Throughput genetic resource for a B. fragilis strain isolated from a UC pouchitis patient. Bile acids limit microbial growth and are altered in abundance in UC pouches, where B. fragilis often blooms. Using this resource, we uncovered pathways and processes that impact B. fragilis fitnessin bile and that may contribute to population expansions during bouts of gut inflammation.