A sequencing batch reactor (SBR) was operated to obtain thiosulfate-utilizing denitrifier cultivated with two types of electron accepter (nitrate and nitrite). Using the microbial biomass obtained from the SBR, batch tests were conducted with different nitrite concentrations (50 and 100 mg-N/L) at pH 7.0, 7.5 and 7.9 to see how free nitrous acid (FNA) negatively works on the thiosulfate-utilizing denitrification of nitrate. The specific denitrification rate (SDR) of nitrate was significantly influenced by pH and FNA. The presence of nitrite caused a remarked decrease of the SDR under low pH conditions, because of the microbiological inhibitory effect of FNA. The minimum SDR was observed when initial nitrite concentration was 100 mg-N/L at pH 7.0. Moreover. the SDR was influenced by the type of electron acceptor used during the SBR operation. Thiosulfate-utilizing denitrifier cultivated with nitrite showed smaller SDR on the thiosulfate-utilizing denitrification of nitrate than those cultivated with nitrate.