This study investigated the effects of the influent inorganic carbon (IC) availability on nitrifying microbial activity of a sequencing batch reactor (SBR) operated for partial-nitritation (PN) at mainstream conditions (temperature of 15 °C and low influent N concentration). By operating the reactor with influent ammonium to inorganic carbon centration ratio (NH4 +-N/IC) of 0.40 ± 0.01 mg N·mg C−1, which results in an excess of IC for full nitritation, the oxidized ammonium fully nitrified, and nitrite accumulation was not reached. Limiting influent IC (influent NH4 +-N/IC of 0.73 ± 0.03 mg N·mg C−1) favored nitrite-oxidizing bacteria (NOB) suppression while nitritation was maintained, leading to nitrite accumulation of over 80%. Low bulk liquid IC reduced pH values and this one also favored the achievement of higher free nitrous acid (FNA) levels, but it did not stop nitrite oxidation to nitrate. PN control was also favored by keeping low bulk oxygen concentration during all the aeration phase (below 0.5 mg O2·L−1). Results obtained by batch assays showed that the ratio between the ammonium (rNH4 + batch) and nitrite oxidation rates (rNO2 − batch) dropped from 0.92 (without IC limitation) to 0.04 (with IC limitation). The results obtained highlight the critical IC role in the microbial interactions of the partial-nitritation process and present new insights in the direction of obtaining NH4 +/NO2 − for mainstream nitrite short-cut nitrogen removal process.