Background: Khat is a psychostimulant plant widely used in Africa and its use has been growing rapidly in Europe and North America. Objectives: We investigated effects of chronic khat (Catha edulis) use on cardiovascular, adrenocortical, and psychological responses to acute stress. Methods: Chronic khat users and nonusers were compared on physiological measures and mood reports in a cross‐sectional, mixed design. Measurements were conducted during 24‐hour ambulatory monitoring and during a laboratory session. A total of 152 participants (58 women) were recruited by flyers posted around Sana’a University campus and the surrounding community in Sana’a, Yemen. Salivary cortisol and self‐report measures were collected during a 24‐hour ambulatory period prior to a lab testing session. In addition, blood pressures (BP), salivary cortisol, and mood measures were assessed during rest and in response to acute mental stress. Results: Khat users exhibited enhanced evening and attenuated morning cortisol levels, reflecting a blunted diurnal pattern of adrenocortical activity compared to nonusers. Khat users reported greater negative affect during the ambulatory period and during the laboratory session. In addition, they exhibited attenuated BP responses to stress. Conclusions and Scientific Significance: These novel results demonstrate altered adrenocortical activity and increased dysphoric mood among khat users. The extent to which these associations are due to effects of chronic khat use per se or instead reflect predisposing risk factors for khat use is yet to be determined. (Am J Addict 2013;22:99–107)