Indole alkaloids from Catharanthus roseus (L.) G. Don were valued for their wide spectrum of pharmaceutical effects. Their biosynthesis is modulated by various abiotic factors including ultraviolet B radiation which induced ATP production and led to accumulation of indole alkaloids. To investigate the regulatory mechanism of secondary metabolism in the leaf mitochondria of C. roseus under ultraviolet B radiation, combinatory study of proteomic and metabolomic analyses of C. roseus was performed. ATP export in the leaves of C. roseus increased under UVB radiation, which required mitochondrial ATP synthase. Proteins related to mitochondrial complexes II/IV and their gene expression levels increased, and those related to mitochondrial complex I decreased. Proteins related to the methylerythritol phosphate pathway, especially geranylgeranyl pyrophosphate synthase, increased under ultraviolet B radiation. Metabolites whose levels changed were mainly alkaloids, organic acids, carbohydrates, phenylpropanoids, and fatty acids, while eight indole alkaloids increased. Integrative analysis of omics data indicated that the metabolism of glutamate and tyrosine was downregulated. These results suggest that ultraviolet B radiation induces dynamic changes in mitochondria in C. roseus, which guarantees ATP production, regulates the flux of the methylerythritol phosphate pathway to the biosynthesis of monoterpene moieties, and leads to accumulation of various indole alkaloids.