A non-volatile semiconductor storage device includes: a semiconductor substrate; a semiconductor layer formed on the semiconductor substrate; a first device isolation/insulation film formed in a trench, the trench formed in the semiconductor layer, with a first direction taken as a longitudinal direction; a device formation region formed by separating the semiconductor layer by the first device isolation/insulation film with the first direction taken as a longitudinal direction; and a memory transistor disposed on the device formation region. The first device isolation/insulation film and the device formation region have an impurity of a first conductivity type. An impurity concentration of the impurity of the first conductivity type in the first device isolation/insulation film is higher than that in the device formation region.