Abstract Objective This study was undertaken to characterize the functional impact of novel SLC12A5 variants in two unrelated patients with early onset developmental and epileptic encephalopathy (DEE) and to investigate the mechanisms underlying KCC2 dysfunction. Methods Clinical, genetic, and functional analyses were performed in two patients (Cases A and B) with DEE. SLC12A5 encodes two KCC2 splice isoforms (KCC2a and KCC2b). Functional effects of the identified variants on KCC2b ion transport, phosphorylation, mRNA processing, and KCC2‐dependent synaptogenesis were assessed using in vitro assays in heterologous expression systems and primary neurons, supported by in silico structural modeling. Results Both patients developed severe neonatal onset DEE characterized by developmental delay, axial hypotonia, extrapyramidal features, and bilateral migratory seizures within 24 h of birth. Both cases resulted in early mortality (Case A at 9 years; Case B at 6 months). Sequencing revealed distinct biallelic compound heterozygous SLC12A5 variants in both individuals, each inherited from one unaffected parent. Functional analyses demonstrated that in Case A, one variant markedly reduced KCC2‐mediated ion transport, whereas the second variant preserved transport activity but exhibited an altered phosphorylation profile at Ser940, located on the intracellular C ‐terminal region. This variant also disrupted wild‐type (WT) KCC2‐dependent excitatory synapse formation in immature rat hippocampal neurons. In Case B, one variant disrupted normal mRNA transcript processing consistent with loss of expression, and the second variant exhibited reduced ion transport activity. Significance These data demonstrate that SLC12A5 ‐related DEE can result from combined impairment of KCC2‐dependent chloride homeostasis and disruption of chloride‐independent KCC2 functions critical for early neuronal development. This work expands the mutational and mechanistic spectrum of SLC12A5 ‐DEE and highlights the importance of KCC2 regulatory roles in early brain development, providing new knowledge and tools for basic research and potential avenues for targeted precision therapies.