The regulation of two-stage caproate fermentation from fruit and vegetable waste (FVW) via anaerobic microbial consortia was investigated in this study. The results showed the highest caproate production achieved 14.9 g/L at the optimal inoculum to substrate ratio (ISR) of 2:1, ethanol to acid ratio (E/A) of 4:1, and pH of 7.5. The caproate yield and selectivity respectively reached 0.62 g/g and 80.8% (as COD). In acidification stage, an appropriate ISR provided a high conversion efficiency and more acetate formation, which was beneficial to caproate biosynthesis. In caproate production stage, chain elongation performance was sensitive to E/A and pH condition. Butyrate became the main by-product at low E/A or acidic conditions, while excessive ethanol or alkaline condition seriously suppressed substrate conversion. The caproate fermentation was dominated by Clostridium kluyveri. Furthermore, caproate formation was uncoupled with Clostridium kluyveri proliferation, which was mainly generated during the middle and late stages of growth.