Introduction <p>Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multisystemic illness, commonly associated with dysregulation of the immune system including reduced cytotoxicity of natural killer (NK) cells and post-exertional neuroimmune exhaustion. Previously, transient receptor potential melastatin 3 (TRPM3) ion channel impairment associated with reduced Ca²⁺ mobilisation in NK cells from ME/CFS patients was reported. To further explore the pathomechanisms involved in ME/CFS, we investigated the downstream impact of TRPM3 ion channel dysfunction on mitochondrial Ca²⁺ mobilisation in NK cells.</p> Method <p>Fluorescence live-cell imaging was used to investigate Ca²⁺ mobilisation in NK cells of (<i>N</i> = 10) ME/CFS, classified using Canadian Consensus Criteria, and (<i>N</i> = 10) healthy control (HC) participants. Cytoplasmic and mitochondrial Ca²⁺ entry was determined using Fluo-8 AM and Rhod-2 AM Ca²⁺ indicators, respectively. The effect of TRPM3 modulation on Ca²⁺ mobilisation ex vivo, was examined using pregnenolone sulfate and ononetin to activate and inhibit the channel, respectively.</p> Results <p>Cytosolic Ca²⁺ influx amplitude and slope were significantly reduced (<i>p</i> &lt; 0.001), with a significantly shorter T_1/2 response (<i>p</i> = 0.001) in ME/CFS compared to HC. Ca²⁺ influx amplitude (<i>p</i> &lt; 0.001) and slope (<i>p</i> &lt; 0.041) into the mitochondria were significantly higher in ME/CFS compared to HC. TRPM3 activation triggered pronounced cytosolic response (<i>P</i> &lt; 0.001) accompanied by mitochondrial Ca²⁺ increase in HC. TRPM3-dependent cytosolic and mitochondrial Ca²⁺ mobilisation (<i>P</i> &lt; 0.015) were significantly reduced with a shorter T_1/2 response (<i>p</i> &lt; 0.02) in ME/CFS compared to HC.</p> Conclusion <p>The results demonstrate that altered TRPM3-mediated cytosolic Ca²⁺ influx may significantly impact Ca²⁺ mobilisation into the mitochondria of people with ME/CFS. Alterations that interfere with the optimal function of Ca²⁺ permeable channels may cumulatively impact downstream signalling, leading to detrimental cellular consequences. Collectively these findings provide an avenue for further studies on the physiological functions of TRPM3 ion channel and its role in ME/CFS.</p>