Small interval LED based naked eye 3D display system is complementary to LCD based naked eye 3D display system for its relative high resolution and large display size. The sub-pixel of LED is point-like and can be arranged in horizontal or vertical directions. In this paper the effect of sub-pixel direction on cross talk of display system is investigated. The Tracepro software is used to analyze crosstalks of two 3D display systems with sub-pixel interval as 1.25 mm and 1.667 mm, respectively. The simulation results shows 3D display systems of different sub-pixel arrangement directions have nearly the same crosstalk. This result is further confirmed by experimentally testing crosstalk of 1.25 mm LED naked eye 3D display system. With 1.25 mm LED naked eye 3D display system, its sub-pixel arrangement direction can be changed freely according to actual requirements.