The electrokinetic motion of a negatively charged spherical particle at an oil-water interface in a microchannel is numerically investigated and analyzed in this paper. A three-dimensional (3D) transient numerical model is developed to simulate the particle electrokinetic motion. The channel wall, the surface of the particle and the oil-water interface are all considered negatively charged. The effects of the direct current (DC) electric field, the zeta potentials of the particle-water interface and the oil-water interface, and the dynamic viscosity ratio of oil to water on the velocity of the particle are studied in this paper. In addition, the influences of the particle size are also discussed. The simulation results show that the micro-particle with a small value of negative zeta potential moves in the same direction of the external electric field. However, if the zeta potential value of the particle-water interface is large enough, the moving direction of the particle is opposite to that of the electric field. The velocity of the particle at the interface increases with the increase in the electric field strength and the particle size, but decreases with the increase in the dynamic viscosity ratio of oil to water, and the absolute value of the negative zeta potentials of both the particle-water interface and the oil-water interface. This work is the first numerical study of the electrokinetic motion of a charged particle at an oil-water interface in a microchannel.