Abstract This project uses methods in geometric analysis to study almost complex manifolds. We introduce the notion of biharmonic almost complex structure on a compact almost Hermitian manifold and study its regularity and existence in dimension four. First, we show that there always exists smooth energy-minimizing biharmonic almost-complex structures for any almost Hermitian four manifold. Then, we study the existence problem where the homotopy class is specified. Given a homotopy class $[\tau ]$ of an almost complex structure, using the fact $\pi _4(S^2)=\mathbb {Z}_2$ , there exists a canonical operation p on the homotopy classes satisfying $p^2=\text {id}$ such that $p([\tau ])$ and $[\tau ]$ have the same first Chern class. We prove that there exists an energy-minimizing biharmonic almost complex structure in the companion homotopy classes $[\tau ]$ and $p([\tau ])$ . Our results show that, When M is simply connected, there exists an energy-minimizing biharmonic almost complex structure in the homotopy classes with the given first Chern class.