Simple SummaryAmmonia (NH3/NH4+) is one of the most common contaminants in the aquaculture environment. Ammonium transporter 1 (AMT1) is a member of the Amt/Mep/Rh family that facilitates movement of ammonia across plasma membranes. However, the role of AMT1 in mollusks remains unclear. In this work, we found that the activation of AMT1 expression in razor clam (Sinonovacula constricta) can be significantly induced under high ammonia exposure, and AMT1 synergizes with Rhesus glycoprotein (Rh) to facilitate ammonia transport. The results will help to better understand the biological functions of AMT1 in razor clam ammonia excretion and improve studies of its molecular mechanism.AbstractAmmonium transporter 1 (AMT1), a member of ammonia (NH3/NH4+) transport proteins, has been found to have ammonia transport activity in plants and microorganisms. However, the functional characteristics and molecular mechanisms of AMT1 in mollusks remain unclear. The razor clam (Sinonovacula constricta) is a suitable model species to explore the molecular mechanism of ammonia excretion because of the high concentration of ambient ammonia it is exposed to in the clam–fish–shrimp polyculture system. Here, the expression ofAMT1inS. constricta(Sc-AMT1) in response to high ammonia (12.85 mmol/L NH4Cl) stress was identified by real-time quantitative PCR (qRT-PCR), Western blotting, RNA interference, and immunofluorescence analysis. Additionally, the association between the SNP_g.15211125A > T linked withSc-AMT1and ammonia tolerance was validated by kompetitive allele-specific PCR (KASP). A significant upregulated expression of Sc-AMT1 was observed during ammonia exposure, and Sc-AMT1 was found to be localized in the flat cells of gill. Moreover, the interference withSc-AMT1significantly upregulated the hemolymph ammonia levels, accompanied by the increased mRNA expression of Rhesus glycoprotein (Rh). Taken together, our findings imply that AMT1 may be a primary contributor to ammonia excretion inS. constricta, which is the basis of their ability to inhabit benthic water with high ammonia levels.