Caffeine, the major purine alkaloid in tea has long been known for its role in plant defense. However, its effect on Colletotrichum gloeosporioides that causes brown blight disease in tea is largely unknown especially under elevated CO2. Here we show that elevated CO2 reduced endogenous caffeine content in tea leaves, but sharply increased susceptibility of tea to C. gloeosporioides. The expression of C. gloeosporioides actin gene was gradually increased during the postinoculation period. In contrast, foliar application of caffeine decreased the C. gloeosporioides-induced necrotic lesions and the expression of C. gloeosporioides actin. Analysis of endogenous jasmonic acid (JA) content revealed that exogenous caffeine could induce JA content under both CO2 conditions in absence of fungal infection; however, in presence of fungal infection, caffeine increased JA content only under elevated CO2. Furthermore, exogenous caffeine enhanced lipoxygenase (LOX) activity and its biosynthetic gene expression under both CO2 conditions, indicating that increased JA biosynthesis via LOX pathway by caffeine might strengthen plant defense only under elevated CO2, while caffeine-induced defense under ambient CO2 might be associated with JA-independent LOX pathway in tea. These results provide novel insights into caffeine-induced plant defense mechanisms that might help to develop an eco-friendly approach for disease control.