Different inoculum and substrate ratio (ISR) was taken to conduct the anaerobic digestion of orange pressing waste (OPR), and the daily production of methane was recorded. The relative abundance of bacteria and archaea in the inoculum and sludge were tested to find out the change before and after anaerobic digestion. And the digested materials were used for the test on the improvement of anaerobic digestion after acclimation of inoculum. The results demonstrated that the cumulative methane production of ISR 8, 6 and 4 were 320.0mL/g, 304.9mL/g and 242.6mL/g, respectively, but that of ISR 2 was only 111.4mL/g after a lag phase of 7d. And the relative abundance of bacterial and archaea community of ISR 2 was quite different from those in other tests. The relative abundance of Bacilli, Methanosaeta and Methanospirillum was decreased to 1.88%, 24.45% and 1.71%, respectively, which may cause the insufficient degradation of the substrate. Although there was no increase in the cumulative methane production in the 2nd and 3rd rounds of anaerobic digestion, the time employed to reach the final methane production was shorter than that in the 1st round of ISR 8 and 6, and the cumulative methane production of the first 4d was 95.75% and 93.40% of the total production in the 3rd rounds. But it was unstable when ISR 4 was taken to conduct the test. In this situation, the improvement of anaerobic digestion would be decided by the source of the inoculum, and no improvement was found in the 2nd and 3rd rounds if the ISR was 2. According to the results, the optimum ISR for the anaerobic digestion of OPR was 6 whether in a test on the biomethane production from different OPR or in a pretreatment unit for the improvement of the methane production, and at least a round of anaerobic digestion was needed for the acclimation of the inoculum.