The purpose of this study was to test the hypothesis that downslope treadmill walking decreases spinal excitability. Soleus H-reflexes were measured in sixteen adults on 3 days. Measurements were taken before and twice after 20 min of treadmill walking at 2.5 mph (starting at 10 and 45 min post). Participants walked on a different slope each day [level (Lv), upslope (Us) or downslope (Ds)]. The tibial nerve was electrically stimulated with a range of intensities to construct the M-response and H-reflex curves. Maximum evoked responses (Hmax and Mmax) and slopes of the ascending limbs (Hslp and Mslp) of the curves were evaluated. Rate-dependent depression (RDD) was measured as the % depression of the H-reflex when measured at a rate of 1.0 Hz versus 0.1 Hz. Heart rate (HR), blood pressure (BP), and ratings of perceived exertion (RPE) were measured during walking. Ds and Lv walking reduced the Hmax/Mmax ratio (P = 0.001 & P = 0.02), although the reduction was larger for Ds walking (29.3 ± 6.2% vs. 6.8 ± 5.2%, P = 0.02). The reduction associated with Ds walking was correlated with physical activity level as measured via questionnaire (r = −0.52, P = 0.04). Us walking caused an increase in the Hslp/Mslp ratio (P = 0.03) and a decrease in RDD (P = 0.04). These changes recovered by 45 min. Exercise HR and BP were highest during Us walking. RPE was greater during Ds and Us walking compared to Lv walking, but did not exceed “Fairly light” for Ds walking. In conclusion, in healthy adults treadmill walking has a short-term effect on soleus H-reflex excitability that is determined by the slope of the treadmill surface.