Abstract Thyroid hormone (TH) is critical for brain development and function. T3 enters neurons through membrane transporters and reaches the cell nucleus where it binds to receptors (TR) to regulate gene transcription. However, neurons also express the type 3 deiodinase (D3), which is located in the cellular and nuclear membranes and inactivates T3. Here, we investigated the fate and biological impact of T3 that enters neurons through axons. Primary cortical neurons were isolated from E16.5 embryos of the TH action indicator (THAI) mice, which were engineered with a TH-responsive transgene where three copies of a T3-responsive element drive a luciferase (Luc) reporter. Neurons were seeded on a microfluidic device consisting of two independent compartments: (i) cellular, where about 70-90,000 cell bodies were located, and (ii) axonal, where a few hundred distal axons were located. Fluidic isolation of the compartments was monitored with Alexa Fluor 594 hydrazide. In the first set of experiments (repeated 3 times), 8-10-day old cultures were incubated for 48h with medium containing 1% charcoal-stripped serum (Tx-medium). Subsequently, 10nM T3 was added to the axonal compartment, and 24h later cell bodies were harvested and Luc mRNA measured by RT-qPCR. There was a 2.4 ± 0.7-fold increase in Luc mRNA levels, but the addition of 2uM Silychristin (MCT8 inhibitor) to the axonal compartment reduced T3 induction of Luc mRNA by 32 ± 4.2%. In the second set of experiments (repeated 3 times), 4.9 ± 2.2pM 125I-T3 (final concentration) was added to the cellular or axonal compartments. Medium was sampled and 125I-T3 and its metabolites were separated/quantified via UPLC linked to a flow scintillation detector. After 72h of adding 125I-T3 to the axonal compartment, about 0.73 % 125I-T3 (0.052 ± 0.025pM) was found in the cellular compartment. In addition, 3,3’-125I-T2 and 125I (0.011 ± 0.003 and 0.052 ± 0.023pM, respectively) were also detected. When 125I-T3 was added to the cellular compartment, about 1.6% 125I-T3 (0.048 ± 0.027pM), no metabolites, was detected in the axonal compartment. Only background radioactivity was detected in the opposing compartment when 125I-T3 was added in the absence of cells. We conclude that T3 can be taken up by neuronal axons, partly via MCT8, and transported retrogradely to the cell nucleus to initiate TH signaling. D3-generated T3 metabolites exit the cell body alongside with small amounts of intact T3. This pathway could explain how D2-generated T3 in tanycytes is taken up by TRH-secreting neurons to mediate negative T3 feedback. Anterograde T3 transport was also detected, the significance of which remains unknown.