Retinal axon specification and growth are critically sensitive to the dosage of numerous signaling molecules and transcription factors. Subtle variations in the expression levels of key molecules may result in a variety of axonal growth anomalies. miR-181a and miR-181b are two eye-enriched microRNAs whose inactivation in medaka fish leads to alterations of the proper establishment of connectivity and function in the visual system. miR-181a/b are fundamental regulators of MAPK signaling and their role in retinal axon growth and specification is just beginning to be elucidated. Here we demonstrate that miR-181a/b are key nodes in the interplay between TGF-β and MAPK/ERK within the functional pathways that control retinal axon specification and growth. Using a variety of in vivo and in vitro approaches in medaka fish, we demonstrate that TGF-β signaling controls the miR-181/ERK regulatory network, which in turn strengthens the TGF-β-mediated regulation of RhoA degradation. Significantly, these data uncover the role of TGF-β signaling in vivo, for the first time, in defining the correct wiring and assembly of functional retina neural circuits and further highlight miR-181a/b as key factors in axon specification and growth.