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TRPM3 belongs to the melastatin sub-family of transient receptor potential (TRPM) cation channels and has been shown to function as a steroid-activated, heat-sensitive calcium ion (Ca²⁺) channel. A missense substitution (p.I65M) in the TRPM3 gene of humans (<i>TRPM3</i>) and mice (<i>Trpm3</i>) has been shown to underlie an inherited form of early-onset, progressive cataract. Here, we model the pathogenetic effects of this cataract-causing mutation using ‘knock-in’ mutant mice and human cell lines. <i>Trpm3</i> and its intron-hosted micro-RNA gene (<i>Mir204</i>) were strongly co-expressed in the lens epithelium and other non-pigmented and pigmented ocular epithelia. Homozygous <i>Trpm3</i>-mutant lenses displayed elevated cytosolic Ca²⁺ levels and an imbalance of sodium (Na⁺) and potassium (K⁺) ions coupled with increased water content. Homozygous <i>TRPM3</i>-mutant human lens epithelial (HLE-B3) cell lines and <i>Trpm3</i>-mutant lenses exhibited increased levels of phosphorylated mitogen-activated protein kinase 1/extracellular signal-regulated kinase 2 (MAPK1/ERK2/p42) and MAPK3/ERK1/p44. Mutant TRPM3-M65 channels displayed an increased sensitivity to external Ca²⁺ concentration and an altered dose response to pregnenolone sulfate (PS) activation. <i>Trpm3</i>-mutant lenses shared the downregulation of genes involved in insulin/peptide secretion and the upregulation of genes involved in Ca²⁺ dynamics. By contrast, <i>Trpm3</i>-deficient lenses did not replicate the pathophysiological changes observed in <i>Trpm3</i>-mutant lenses. Collectively, our data suggest that a cataract-causing substitution in the TRPM3 cation channel elicits a deleterious gain-of-function rather than a loss-of-function mechanism in the lens.