We introduce the notion of emph{biharmonic almost complex structure} on a compact almost Hermitian manifold and we study its regularity and existence in dimension four. First we show that there always exist smooth energy-minimizing biharmonic almost complex structures for any almost Hermitian structure on a compact almost complex four manifold, and all energy-minimizers form a compact set. Then we study the existence problem when the homotopy class of an almost complex structure is specified. We obtain existence of energy-minimizing biharmonic almost complex structures which depends on the topology of $M^4$. When $M$ is simply-connected and non-spin, then for each homotopy class which is uniquely determined by its first Chern class, there exists an energy-minimizing biharmonic almost complex structure. When $M$ is simply-connected and spin, for each first Chern class, there are exactly two homotopy classes corresponding to the first Chern class. Given a homotopy class $[tau]$ of an almost complex structure, there exists a canonical operation on the homotopy classes $p$ 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 (at least) one of the two homotopy classes, $[tau]$ and $p([tau])$. In general if $M$ is not necessarily simply-connected, we prove that there exists an energy-minimizing biharmonic almost complex structure in (at least) one of the two homotopy classes $[tau]$ and $p([tau])$. The study of biharmonic almost complex structures should have many applications, in particular for the smooth topology of the underlying almost complex four manifold. We briefly discuss an approach by considering the moduli space of biharmonic almost complex structures and propose a conjecture.