Primary Nitrite Maximum (PNM) is ubiquitously found at the bottom of the euphotic zone in stratified global oceans. Due to complex source-sink processes of nitrite involving interactions between photoautotrophs and chemoautotrophic microorganisms, its formation mechanism remains inconclusive. Previous isotope-tracing evidence suggests that nitrite in the PNM is mainly regulated by ammonia oxidation (source) mediated by ammonia-oxidizing archaea (AOA) and nitrite oxidation (sink) mediated by the nitrite-oxidizing bacteria (NOB). However, previous studies have been constrained by technical limitations, focusing solely on bottom-up controls (e.g., light and substrates), while top-down controls (zooplankton grazing on chemoautotrophic AOA and NOB) have remained unquantifiable. Based on the developed first methodology enabling simultaneous quantification of in situ grazing rates and growth rates of both AOA and NOB in marine environments, we conducted three cruises and found that the grazing rates of AOA and NOB were both rapid at the PNM in the Northwest Pacific and in the South China Sea. Specifically, grazing on NOB is more rapid than AOA, while the proportion of growth consumed by grazing-induced mortality (m/μ) of them shows no significant difference. Our study suggested that grazing plays a previously unanticipated important role at the base of euphotic zone. Through quantitatively evaluating the regulation of nitrite concentration by grazing, we indicated that top-down control plays a critical role at the formation of PNM in the tropical and subtropical stratified ocean, delivering new insights into the underlying mechanisms of this classic oceanographic issue.