OBJECTIVE: KCNB1 encodes an α-subunit of the delayed-rectifier voltage-dependent potassium channel K2.1. De novo pathogenic variants of KCNB1 have been linked to developmental and epileptic encephalopathies (DEEs), diagnosed in early childhood and sharing limited treatment options. Loss of function (LOF) of KCNB1 has been proposed as the pathogenic mechanism in these disorders. Here, we aim to characterize a knockout zebrafish line targeting kcnb1 (kcnb1 and kcnb1) for investigating DEEs. METHODS: This study presents the phenotypic analysis of a kcnb1 knockout zebrafish model, obtained by CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) mutagenesis. Through a combination of immunohistochemistry, behavioral assays, electrophysiological recordings, and neurotransmitter quantifications, we have characterized the expression, function, and impact of this kcnb1 LOF model at early stages of development. RESULTS: In wild-type (WT) larval zebrafish, kcnb1 was found in various regions of the central nervous system and in diverse cell subtypes including neurons, oligodendrocytes, and microglial cells. Both kcnb1 and kcnb1 zebrafish displayed impaired swimming behavior and "epilepsy-like" features that persisted through embryonic and larval development, with variable severity, which was restored by the human K2.1 WT DNA. When exposed to the chemoconvulsant pentylenetetrazol (PTZ), both knockout models showed elevated locomotor activity. In addition, PTZ-exposed kcnb1 larvae exhibited increased bdnf mRNA expression and higher c-Fos fluorescence intensity in cells of the telencephalon. This same model presents spontaneous and provoked epileptiform-like electrographic activity associated with γ-aminobutyric acid dysregulation, whereas the brain anatomy and neuronal circuit organization remained unaffected. SIGNIFICANCE: We conclude that kcnb1 knockout in zebrafish leads to early onset phenotypic features reminiscent of DEEs, affecting neuronal functions and primarily inhibitory pathways in developing embryonic and larval brains. This study highlights the relevance of this model for investigating developmental neuronal signaling pathways in KCNB1-related DEEs.