Magnetic anisotropy has significant importance in both designing high-efficiency magnetic devices and understanding noncollinear spin textures. Here, La0.67Sr0.33MnO3 layers are epitaxially fabricated on the (001)-, (110)-, and (111)-orientated SrTiO3 substrates to manipulate magnetic anisotropy. While the [11¯0] axis is the in-plane magnetic easy axis for the (001) La0.67Sr0.33MnO3 layer, it becomes the hard axis for the (110) and (111) samples. Interestingly, the (110) samples exhibit a great enhancement in magnetic anisotropy energy, reaching at 6.1 × 105 erg/cm3 that is 1–2 orders of magnitude higher than ones in (001) and (111) films. It is also noted that such improved magnetic anisotropy of (110) samples is maintained up to 30 nm. Those observations can be interpreted in terms of the orientation-modified orbital symmetry. While the energetically favored 3dx2−y2 orbital and degenerated eg orbitals drive the two-dimensional and bulk-like ferromagnetism in (001) and (111) La0.67Sr0.33MnO3 layers, respectively, the 3d3z2−r2 orbital provides the lowest orbital symmetry and results in the highest magnetic anisotropy energy in (110) samples. Meanwhile, this model can also explain the thickness-dependent Curie temperatures with different epitaxial orientations, deepening the understanding of the complex oxide heterostructures for magnetic modulations.