A key pathological hallmark of neurodegenerative diseases is the abnormal accumulation of amyloid aggregates in cells, which leads to neuronal malfunction and cell death (1). It was reported that liquid–liquid phase separation (LLPS) is closely regarded as an initial stage of amyloid aggregation (2), such as Tau in Alzheimer’s disease (AD). Tau is a microtubule-associated protein, which undergoes the LLPS in neurons. In physiological conditions, droplets of Tau participate in tubulin assembly in dynamics, spatially regulating the functions of microtubules (3). While under pathological conditions, the droplets of Tau gradually transform into a solid-like state, eventually forming aggregates and deposits (4). Among various factors that manipulate the LLPS behavior of Tau, a considerable factor is post-translational modifications (PTMs) , such as phosphorylation and glycation. In addition, 14-3-3 protein that can interact with Tau is a potential regulator of Tau LLPS. Here, we elucidate that phosphorylation and glycosylation regulate the behavior of TAU and 14-3 co-phase separation and consequently affect subsequent microtubule assembly and fiber growth.