INTRODUCTION: Human tau protein, composed of six brain-specific isoforms, is a major driver of Alzheimer's disease (AD). The role of its isoforms however remains unclear and human AD models are scarce. METHODS: We generated human MAPT- (tau-) knockout (KO) induced pluripotent stem cells (iPSC) using CRISPR/Cas9, differentiated these into glutamatergic neurons, and assessed isoform-specific functions of tau in these neurons. We used omic- approaches, live-cell imaging, subcompartmental analysis, and lentivirus-based reintroduction of specific tau isoforms to investigate isoform-mediated neuronal dysfunction in an AD model. RESULTS: Tau KO human iPSC-derived neurons showed decreased neurite outgrowth and axon initial segment length and, notably, resisted amyloid beta oligomer (AβO)-induced neuronal activity reduction. Introducing the 1N4R-tau isoform, but not other isoforms, confers AβO vulnerability and increases KxGS phosphorylation of tau, without altering neuronal activity or microtubule modifications. DISCUSSION: While tau KO impacts neuronal development and activity, tau-KO also confers resistance against AβO insult. 1N4R-tau likely mediates AβO-induced and phosphorylated tau toxicity, representing a novel prime therapeutic target for AD. HIGHLIGHTS: Tau knockout alters neurite growth and axon initial segment formation in human neurons. Tau isoforms show differential axonal localization in human neurons. Tau depletion protects against amyloid beta oligomer (AβO)-mediated neurotoxicity. 1N4R tau mediates AβO-induced toxicity in human neurons.