Spinal cord injury (SCI) is a debilitating condition within the nervous system with a high disability rate and substantial economic burden. The functional recovery following SCI is enhanced by moderate levels of autophagy but hindered when autophagy becomes excessive. Galectin-3 (GAL3) has been recognized as an autophagy regulator; however, its role in SCI and its associated mechanism are largely unknown. Here we report that GAL3 was increased in spinal neurons and serum in SCI rats, and knockdown or inhibition of GAL3 promoted motor function recovery. The bioinformatics analysis showed that GAL3 is closely related to programmed cell death after SCI. Indeed, the knockdown of GAL3 resulted in a decrease in autophagy markers ATG7 and LC3 II/I ratio, along with an increase in P62 expression. Furthermore, GAL3 and cell division cycle 42 (CDC42) exhibited close associations with neuronal autophagy. Injection of CDC42 inhibitor ML141 effectively reduced GAL3-mediated enhancement of neuronal autophagy. Additionally, CDC42 was increased in spinal neurons post-SCI, and administration of ML141 decreased the expression of autophagy markers and improved motor function recovery. Importantly, elevated levels of GAL3 and CDC42 were observed in the serum of SCI patients. These findings suggest a potential interplay between GAL3 and CDC42 in regulating neuronal autophagy after SCI and indicate the therapeutic potential of targeting this pathway for enhancing recovery from spinal cord injuries. Overall design: Total RNA was extracted from the spinal cord neurons of neonatal SD rats using Trizol reagent for subsequent sequencing. Sequencing was performed on an Illumina high-throughput sequencing platform (HiSeqTM2500/4000). There were a total of six samples, all of which were treated with high concentrations of glutamate to simulate a neuronal injury model. Among these, three samples were from neurons in the transfection control group, designated as Glu_1, Glu_2, and Glu_3, respectively. The other three samples were from neurons with GAL3 knocked out, designated as Glu_L_1, Glu_L_2, and Glu_L_3, respectively.