The aim of this study was to compare muscle activation and cardio-respiratory response during ergometer and on-water rowing. Nine internationally competitive rowers (five Olympic Games medal winners, age 25.6 ± 4.8 years) were requested to perform a 1,000 m race simulation test in the two conditions. Surface electromyographic (sEMG) signals from trapezius superior (TRS), latissimus dorsi (LD), biceps brachii (BB), rectus femoris (RF), vastus medialis (VAM), vastus lateralis (VAL), biceps femoris (BF) and tibialis anterior (TA) muscles were recorded continuously during the tests together with other cardio-respiratory parameters: heart rate (HR), ventilation (VE), oxygen consumption (VO₂). On-water, subjects covered the same distance in a longer time (218.4 ± 3.8 s vs. 178.1 ± 5.6 s during ergometer test). TRS, LD, BB, RF, VAM and VAL muscle activation on-water was lower than off-water during the rowing race. VO₂ and VE responses were similar between the two conditions even if the time to complete the 1,000 m race simulation test was higher on-water. The results indicate that for most of the analyzed muscles EMG activation on the ergometer is higher than on-water with the maximal activity at the beginning of the on-water test due reasonably to overcome the forces opposing the forward motion, while the ergometer task elicited increasing muscle activation over time. The present data may be considered by coaches when choosing a rowing ergometer in substitution for the training on-water or when relying on the indoor tests to select the crew.