KCC2 is a neuronal-specific chloride extruder that acts to maintain a low intraneuronal chloride concentration, which is critical for fast synaptic inhibition through the GABAA receptor channels. In both humans and mice, KCC2 encoding gene is translated into two isoforms (KCC2a and KCC2b) that differ in their N-terminal domain. While KCC2b is strongly upregulated after birth, KCC2a remains very weakly expressed even in mature neurons. Despite its low expression, KCC2a has been shown to be essential for respiratory behaviour at birth, but the specific function of KCC2a in mature neurons has not yet been investigated.Here we investigated the expression pattern and function of KCC2a in mature neurons in primary hippocampal cultures. Using recombinant flag-tagged KCC2 isoforms, we show that KCC2a has lower dendritic membrane expression compared to KCC2b. However, KCC2a and KCC2b form similar membrane-inserted clusters with comparable localization near glutamatergic and GABAergic synapses. Using a KCC2a-specific short hairpin RNA interference approach, we also show that despite very low expression in hippocampal neurons, KCC2a exerts a strong control over KCC2b expression and function, regulating both its degradation and membrane stability.Thus, KCC2a knockdown impairs KCC2b membrane trafficking and recycling, resulting in reduced membrane expression and impaired chloride transport function. In addition, we demonstrate that KCC2a is required for the regulation of KCC2b membrane expression by the WNK-SPAK/OSR1 pathway. Collectively, our results reveal an unexpected role for the KCC2a isoform in regulating KCC2b expression and function in mature hippocampal neurons, which may be essential for controlling neuronal chloride homeostasis.