A three-dimensional memory device and a method of manufacturing a three-dimensional memory device are provided. The method includes providing a precursor structure including a substrate, a multi-layered stack, a plurality of vertical channel pillars and a barrier structure. A first slit and a second slit are then formed in the multi-layered stack and the substrate along a first direction, in which the first slit and the second slit have a pitch between thereof, and the second slit cuts the barrier structure. A portion of the second insulating layers is then replaced with a plurality of conductive layers. A first slit structure and a second slit structure are then formed in the first slit and the second slit, in which the first slit structure and the second slit structure separate the vertical channel pillars in a second direction that is different from the first direction.