Infection by Sclerotium rolfsii will cause serious disease and lead to significant economic losses in chili pepper. In this study, the response of pepper during S. rolfsii infection was explored by electron microscopy, physiological determination and integrated proteome and metabolome analyses. Our results showed that the stomata of pepper stems were important portals for S. rolfsii infection. The plant cell morphology was significantly changed at the time of the fungal hyphae just contacting (T1) or surrounding (T2) the pepper. The chlorophyll, carotenoid, and MDA contents and the activities of POD, SOD, and CAT were markedly upregulated at T1 and T2. Approximately 4129 proteins and 823 metabolites were clearly identified in proteome and metabolome analyses, respectively. A change in 396 proteins and 54 metabolites in pepper stem tissues was observed at T1 compared with 438 proteins and 53 metabolites at T2. The proteins and metabolites related to photosynthesis and antioxidant systems in chloroplasts and mitochondria were disproportionally affected by S. rolfsii infection, impacting carbohydrate and amino acid metabolism. This study provided new insights into the response mechanism in pepper stems during S. rolfsii infection, which can guide future work on fungal disease resistance breeding in pepper.