In this study, we used a two-stage experiment in order to investigate the effect of the inoculation with elemental sulfur-based autotrophic denitrification (S0-SADN) sludge on the start-up characteristics of Anaerobic ammonia oxidation (ANAMMOX). In the first stage, we attempted direct enrichment with an elemental sulfur and pyrite filler in a S0-SADN reactor, which retained stable operation, and adjusted the nitrogen source components of the influent at different times. In the second stage, we replaced the original filler with Kaldnes filler, and set the influent component to be divided into NH4+-N and NO2−-N. The ANAMMOX process could not be started in the 80-day S0-SADN stage despite the 0.8% abundance of Candidatus Kuenenia; however, after changing the original filler, the reactor showed obvious ANAMMOX reaction characteristics after day 44, and under the condition of an influent TIN load of 0.36 kg(m3·d)−1, the reactor TIN removal rate was stable at more than 80% after day 55. The main ANAMMOX bacteria in the reactor were Candidatus Brocadia (1.08%) and Candidatus Kuenenia (0.96%). The results show that it is feasible to initiate the ANAMMOX process by inoculating the S0-SADN sludge; however, it is not suitable to start the ANAMMOX and the stable operation of the S0-SADN simultaneously. The ANAMMOX process can be started first under the condition of no sulfur source, which takes little time. After initiating the ANAMMOX process, the coupling S0-SADN process can be re-considered given an excessive accumulation of S0-SADN bacteria in the system.