Marine ecosystems are prone to hyposalinity events due to rainfall and freshwater inflow, which has led marine bivalves to develop mechanisms to adapt to salinity fluctuations over a long period of evolution. This study conducted a comprehensive assessment to examine the physiological impact of a low salinity event on Pinctada maxima, which included measure- ments of haemolymph electrolytes, haemocytes functions, and non-targeted metabolomics analyses. A total of 92 metabolites were detected in the gill tissue of P. maxima. Six hours after exposure, low salinity stress impacted five metabolomic pathways, with succinic acid and alanine being highly upregulated. However, the electrolyte and the measured immune response were unaffected at that sampling time. After 24 h of exposure to low salinity conditions, a significant decrease in the concentrations of key electrolytes in the haemolymph was observed, including Na+, Cl−, K+, and Ca2+. Furthermore, the immune responses of the pearl oysters were affected by hypo-salinity stress. Additionally, the total haemocyte count decreased significantly, and some important immune functions, such as the phagocytosis capacity and the intracellular oxidative activity were suppressed. After 72 h of exposure to low salinity, an increase in alanine and succinic acid levels indicated the onset of anaerobic metabolism. The prolonged hyposalinity exposure (72 h) impacted nine metabolomic pathways. The downregulation of adenosine suggested the impairment of the cyclic adenosine monophosphate (cAMP) pathway, which inhibits ambient inorganic ions entering the gill cells. This study identified potential metabolic biomarkers such as succinic acid in oysters exposed to hyposalinity stress.