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Background African trypanosomosis, primarily transmitted by tsetse flies, remains a serious public health and economic challenge in sub-Saharan Africa. Interventions employing natural repellents from non-preferred hosts of tsetse flies represent a promising management approach. Although zebras have been identified as non-preferred hosts of tsetse flies, the basis for this repellency is poorly understood. We hypothesized that zebra skin odors contribute to their avoidance by tsetse flies. Methodology/Principal findings We evaluated the effect of crude zebra skin odors on catches of wild savannah tsetse flies (Glossina pallidipes Austen, 1903) using unbaited Ngu traps compared to the traps baited with two known tsetse fly management chemicals; a repellent blend derived from waterbuck odor, WRC (comprising geranylacetone, guaiacol, pentanoic acid and δ-octalactone), and an attractant comprising cow urine and acetone, in a series of Latin square-designed experiments. Coupled gas chromatography-electroantennographic detection (GC/EAD) and GC-mass spectrometry (GC/MS) analyses of zebra skin odors identified seven electrophysiologically-active components; 6-methyl-5-hepten-2-one, acetophenone, geranylacetone, heptanal, octanal, nonanal and decanal, which were tested in blends and singly for repellency to tsetse flies when combined with Ngu traps baited with cow urine and acetone in field trials. The crude zebra skin odors and a seven-component blend of the EAD-active components, formulated in their natural ratio of occurrence in zebra skin odor, significantly reduced catches of G. pallidipesby 66.7% and 48.9% respectively, and compared favorably with the repellency of WRC (58.1%– 59.2%). Repellency of the seven-component blend was attributed to the presence of the three ketones 6-methyl-5-hepten-2-one, acetophenone and geranylacetone, which when in a blend caused a 62.7% reduction in trap catch of G. pallidipes. Conclusions/Significance Our findings reveal fundamental insights into tsetse fly ecology and the allomonal effect of zebra skin odor, and potential integration of the three-component ketone blend into the management toolkit for tsetse and African trypanosomosis control., Author summary The use of repellents from non-preferred hosts represents an innovative approach to control animal African trypanosomosis by limiting contact between tsetse flies and livestock. Although zebras are non-preferred hosts, the possible chemical basis of their avoidance by tsetse flies is unknown. We hypothesized that certain chemical components of zebra skin odor play a role in their avoidance. We tested this hypothesis by screening crude zebra skin odor for repellency, identifying the chemicals detected by tsetse flies (G. pallidipes) in the skin odor matrix of zebra, and establishing the specific chemicals eliciting repellency in field trials. We identified a three-component blend as contributing to the repellency of the crude zebra skin odor. Repellency of the crude zebra skin odor and the three-component repellent blend was comparable to the known tsetse repellent, WRC (waterbuck repellent compounds), derived from waterbuck skin odor. Our study shows that odors play a role in the avoidance behavior of tsetse flies to zebras. The three-component repellent blend identified in zebra skin odor provides a new effective tool in the management of tsetse flies and trypanosomosis.