Ammonia nitrogen has been a potential menace to aquatic animals along the coastline of China. Presently, the toxicological effects of ammonia nitrogen were mainly concentrated on fishes, while little attention has been paid to molluscs. In this study, the clam Ruditapes philippinarum was used as the target animal to investigate the toxic effects of ammonia nitrogen. Our results showed that ammonia exposure could significantly reduce the integrity of lysosomes in a dose-dependent manner. Metabolite analysis revealed that exposure doses and duration time of ammonia nitrogen could affect the variation profiles of gill metabolites. In detail, branched chain amino acids, glutamate, choline and phosphocholine were significantly decreased after a one-day exposure. Inosine and phenylalanine were found significantly increased and ATP was decreased after a three-day exposure. The changes of metabolites implied that metabolisms of muscle element, neurotransmission and cell apoptosis of gill tissues would be affected by ammonia exposure. Such inferences were supported by the diminished muscle element, decreased concentrations of catecholamines and increased apoptosis rates, respectively. Therefore, we take advantage of metabolomics integrated with conventional biological assays to find out that ammonia exposure could cause lysosome instability, metabolic disturbance, aberrant gill structures and changes to neurotransmitters, and would result in mollusk gill dysfunction in feeding, respiration and immunity.