Abstract: Graphene has a wide range of potential applications in micro/nano electromechanical systems (MEMS/NEMS) due to its excellent lubrication properties. In this paper, the nanofriction anisotropy of graphene surface with different thicknesses was investigated as the tip slides on graphene surface along with different directions by atomic force microscopy (AFM) and high-precision rotating table. The results showed that due to the extremely low out-of-plane stiffness of graphene with a thickness of 3.6 nm, a strong friction strengthening effect was generated in the stick-slip characteristic experiment after the tip sliding on the graphene surface. Then, graphene produced a great out-of-plane deformation and formed a puckering, which increased the contact area between the tip and graphene and thus increased the friction force. As a result, the nanofriction on the surface of graphene exhibited significant anisotropy with a large friction anisotropy ratio. The friction anisotropy ratio was the maximum friction in different angles divided by the minimum friction, and the great out-of-plane deformation leaded to the non-periodic friction of the tip along with all directions. The stiffness of graphene increased with the increase of the thickness leading to an enhanced effect of stick-slip characteristics experiment of small friction due to the weakened out-off-plane deformation, but the out-of-plane deformation did not disappear completely. The zigzag lattice spacing decreased and the armchair lattice spacing increased, which began to close two lattice structures of graphene itself, indicating that the lattice structure began to dominate friction anisotropy. Furthermore, the effect of out-of-plane deformation and lattice structure resulted in periodic anisotropy of 180° and 90° of nanofriction on the surface of graphene with 7.5 nm and 14.7 nm thickness. Due to the large out-of-plane stiffness of graphene with a thickness of 24.5 nm, the lateral force tended to be stable in the stick-slip characteristic experiment, and the friction strengthening effect disappeared, indicating the out-of-plane deformation disappears. Moreover, the spacing between the zigzag lattice and the armchair lattice was almost consistent with the spacing of graphene itself. The results indicated that the frictional anisotropy was inhibited by the increased thickness of graphene and the frictional anisotropy dominated by the lattice structure orientation, resulting in a 60° periodic anisotropy of the surface nanofriction. Also, the friction along the armchair lattice orientation was greater than that along with the sawtooth lattice orientation. The coupling of structural anisotropy and thickness was the main factor of nanofriction anisotropy on the graphene surface. This work revealed the relationship between the different thicknesses of graphene and the anisotropy of surface nanofriction, which provided a theoretical basis for the design and application of graphene in micro and nanomechanical and electrical devices lubrication.