Recent literature has highlighted altered spinal-reflex excitability following acute lateral ankle sprain (ALAS), yet there is little information on the conduction velocity of spinal reflex pathways (CV-SRP) in these patients. Therefore, we aimed to investigate the effects of ALAS on the CV-SRP. We employed a cross-sectional study with two groups: ALAS (n = 30) and healthy controls (n = 30). The CV-SRP of the soleus, fibularis longus, and tibialis anterior was assessed using the H-index method. As secondary outcomes, H-reflex and M-wave latencies were assessed as well as acute symptoms including ankle swelling, pain, and self-reported ankle function. Separate group-by-limb ANOVA with repeated measures revealed a significant interaction for soleus CV-SRP (ppp > 0.05). A further correlation analysis showed a significant relationship between CV-SRP and acute symptoms, including ankle swelling (r = −0.37, p = 0.048) and self-reported ankle function (r = 0.44, p = 0.017) in ALAS patients. These results suggest a disrupted functionality of the afferent pathway and/or synaptic transmission following ALAS. Level of Evidence: 4. View Full-Text ►▼ Show Figures This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.