Abstract Acute aerobic exercise (AAE) can modulate primary motor cortex (M1) excitability. To date, studies evaluating the effects of AAE on M1 excitability have focused almost exclusively on concentric cycling. Interestingly, eccentric cycling increases frontal-parietal brain activation more than concentric cycling. Critically, we recently found eccentric AAE enhances motor learning more than concentric AAE. Yet, the M1 excitability mechanisms underlying these effects remain unknown. Thus, the objective of this study was to evaluate the effect of eccentric cycling AAE on M1 excitability using transcranial magnetic stimulation (TMS). Thirty adults performed three 20 min-conditions: i) eccentric cycling AAE, ii) concentric cycling AAE, and iii) rest. Cycling AAE was carried out at a workload corresponding to 70% of peak heart rate (%HRpeak) measured during concentric incremental cycling exercise. TMS assessments were conducted before (Pre), immediately (Post0) and 20 minutes after (Post20) AAE/rest to evaluate changes in corticospinal excitability (CSE) and short interval-intracortical inhibition (SICI). We found CSE increased and SICI decreased at Post20 following eccentric and concentric cycling AAE compared to rest. Also, %HRpeak, muscle pain and perceived effort were lower during eccentric cycling AAE compared to concentric cycling AAE. Our results showed that eccentric cycling impacted M1 excitability change to a comparable degree as concentric cycling, while requiring less cardiovascular response, eliciting less muscle pain and lower perceived effort. Taken together, our results suggest that eccentric cycling AAE may be a valuable intervention to modulate M1 excitability for populations with limited cardiovascular capacity and have potential implications in clinical and sports-related contexts. New and Noteworthy This study demonstrates that both eccentric and concentric cycling exercise enhance corticospinal excitability and reduce short-interval intracortical inhibition. Additionally, eccentric cycling elicited significantly lower cardiovascular and perceptual responses compared to concentric cycling. These findings suggest that eccentric cycling may be a useful intervention for individuals with limited exercise capacity, such as clinical or aging populations.