Transient receptor potential (TRP) ion channels possess high permeability for calcium and upon activation facilitate cell function in non-excitable cells including lymphocytes. Calcium is in part responsible for cell differentiation, proliferation, programmed cell death, and transcriptional events. There is evidence that TRP melastatin 3 (TRPM3) plays a role in impaired natural killer (NK) cell activity consistently reported in Myalgic Encephalomyelitis (ME). ME is a severe and elusive multisystemic illness hallmarked by post-exertional neuroimmune exhaustion accompanied by symptoms categorised as neurological, immunological, gastrointestinal, cardiovascular and endocrinological. People with ME (n=125), according to the Canadian Consensus Criteria for ME, and non-fatigue controls (NFCs, n=128) were allocated to a series of studies. Whole blood was collected, and NK cells were isolated from using immunomagnetic negative selection. Single nucleotide polymorphisms (SNPs) were examined for TRP ion channel genes via Agena Bioscience iPlex Gold assay. Surface expression of TRPM3 protein was determined using flow cytometry. TRPM3-dependent cytosolic and mitochondrial calcium influx was determined using flow cytometry and live cell immunofluorescent imaging. TRPM3 current amplitude was assessed using whole-cell patch clamp. Five SNPs were identified associated with TRPM3, other SNPs identified were associated with TRPM8, TRPC2 (canonical) and TRPC4. Surface expression of TRPM3 was significantly reduced on NK cells of ME compared with NFCs (p<0.05). Cytosolic and mitochondrial calcium influx via TRPM3 was significantly reduced in ME compared with NFCs (p<0.05). TRPM3 current amplitude was significantly reduced in ME compared with NFCs (p<0.05). The novel identification of TRPM3 dysfunction in ME at genetic, protein and functional levels provides a potential biomarker for the pathomechanism of ME. TRPM3 dysfunction results in altered calcium mobilisation essential for NK cell effector functions. Given the expression of TRPM3 is reported in multiple cell types, TRPM3 dysfunction may account for the multisystemic clinical presentation of ME.