The anticancer drug methotrexate (MTX) was successfully incorporated into the layered ceramic nanoparticles of layered double hydroxides (LDHs). The prepared MTX-LDH two-dimensional nanoassemblies showed enhanced chemical and thermal stability of intercalated MTX molecules through the effective electrostatic interaction with ceramic LDH layers. And the surface charge of MTX-LDHs was determined to be highly positive whereas that of MTX is fairly negative. The anticancer effect of MTX-LDH compared with MTX was evaluated in the human osteosarcoma cell culture line SaOS-2, showing much faster drug efficacy with low concentration. The time-dependent cellular uptake of MTX determined by high performance liquid chromatography showed the much enhanced cellular drug concentration of MTX-LDH treated cancer cells than the MTX treated ones. The particle size of MTX-LDH nanoassemblies was estimated in various solutions such as saline, phosphate saline, and RPMI 1640 cell culture medium, showing that the hydrodynamic size maintains at ~100 nm, regardless of the suspension media. The positive surface charge and ~100 nm size of MTX-LDHs is considered to be the major factor in the high cellular uptake and enhanced drug efficacy.