Rehabilitating walking in ambulatory patients post-stroke, with training that is safe, task-specific, intensive, and of sufficient duration, can be challenging. Some challenges can be met by using body-weight-supported treadmill training (BWSTT). However, it is not known to what degree walking characteristics are similar during BWSTT and overground walking. In addition, important questions regarding the training protocol of BWSTT remain unanswered, such as how proportion of body-weight support (BWS) and walking speed affect walking characteristics during training. The objective was therefore to investigate if and how kinematic walking characteristics are different between overground walking and treadmill walking with BWS in ambulatory patients post-stroke, and the acute response of altering walking speed and percent BWS during treadmill walking with BWS. A cross-sectional repeated-measures design was used. Ambulating patients post-stroke walked in slow, preferred, and fast walking speed overground and at comparable speeds on the treadmill with 20% and 40% BWS. Kinematic walking characteristics were obtained using a kinematic sensor attached over the lower back. Forty-four patients completed the protocol. Kinematic walking characteristics were similar during treadmill walking with BWS, compared to walking overground. During treadmill walking, choice of walking speed had greater impact on kinematic walking characteristics than proportion of BWS. Faster walking speeds tended to affect the kinematic walking characteristics positively. This implies that in order to train safely and with sufficient intensity and duration, therapists may choose to include BWSTT in walking rehabilitation also for ambulatory patients post-stroke without aggravating gait pattern during training.