Dent disease is a rare renal tubular disease with X-linked recessive inheritance characterized by low molecular weight proteinuria (LMWP), hypercalciuria, and nephrocalcinosis. Mutations disrupting the 2Cl-/1H+ exchange activity of chloride voltage-gated channel 5 (CLCN5) have been causally linked to the most common form, Dent disease 1 (DD1), although the pathophysiological mechanisms remain unclear. Here, we conducted the whole exome capture sequencing and bioinformatics analysis within our DD1 cohort to identify two novel causal mutations in CLCN5 (c.749 G > A, p. G250D, c.829 A > C, p. T277P). Molecular dynamics simulations of ClC-5 homology model suggested that these mutations potentially may induce structural changes, destabilizing ClC-5. Overexpression of variants in vitro revealed aberrant subcellular localization in the endoplasmic reticulum (ER), significant accumulation of insoluble aggregates, and disrupted ion transport function in voltage clamp recordings. Moreover, human kidney-2 (HK-2) cells overexpressing either G250D or T277P displayed higher cell-substrate adhesion, migration capability but reduced endocytic function, as well as substantially altered transcriptomic profiles with G250D resulting in stronger deleterious effects. These cumulative findings supported pathogenic role of these ClC-5 mutations in DD1 and suggested a cellular mechanism for disrupted renal function in Dent disease patients, as well as a potential target for diagnostic biomarker or therapeutic strategy development.