Myelin, critical for the correct function of the nervous system, is organized in different patterns that can include long non-myelinated axonal segments. How myelin patterning is regulated remains unexplained. The carbohydrate-binding protein galectin-4 (Gal-4) influences oligodendrocyte differentiation in vitro and is associated with non-myelinable axon segments (NMS) in cultured neurons. In consequence, Gal-4 has been proposed as a myelin patterning regulator, although no in vivo studies have corroborated this hypothesis. We used Gal-4-deficient mice (Lgals4-KO) to study the role of Gal-4 in cortical myelination in vivo. We show that cultured neurons of Lgals4-KO mice form NMS that are regulated as in control neurons. In addition, oligodendrocyte/myelin markers expression measured by biochemical and immunochemical means, and cortical myelin microstructure studied by in-depth image analysis appear unaltered in these animals. Consistently, myelin displays an essentially normal function assessed by in vivo electrophysiology and locomotion analyses. In conclusion, cortical myelin of Lgals4-KO mice does not show any significant defect in composition, organization or function, pointing to a negligible role of Gal-4 in myelination in vivo or, as discussed, to unknown mechanisms that compensate its absence.