AAn experiment was conducted to study the effects of a soilless substrate composed mainly of fermentation bed paddings on the growth and yield of pepper and the effects of continuous pepper cultivation on physicochemical and microbiological properties of the soilless substrate, and the reuse effects of the soilless substrate were also studied. The results showed that there were no significant differences between soilless substrate cultivation and soil cultivation in plant height, root length, plant fresh weight and yield. Besides, the yield of pepper was not affected by planting it with old substrate in the 2nd pepper stubble compared with the 1st stubble. The physicochemical properties of soilless substrate were significantly influenced by pepper planting, particularly for the values of electrical conductivity (EC), available nitrogen and available potassium, which were reduced by 81.17%, 92.15% and 85.16%, respectively. The physicochemical properties of soilless substrate were not significantly changed in the 2nd pepper stubble compared with the 1st stubble. The results of Real-Time PCR analysis showed that the population of bacteria in soilless substrate was not significantly affected between the 1st and 2nd pepper stubbles, while the population of fungi was significantly decreased, but was not significantly changed between the 1st and 2nd pepper stubbles. The results of PCR-DGGE analysis showed that the community structures of bacteria and fungi in soilless substrate was significantly influenced by the 1st pepper planting, and fungal diversity was significantly enhanced. However, neither the community structures nor the diversities of bacteria and fungi were influenced significantly between the 1st and 2nd pepper stubbles.