Objective: Stair climbing is considered a good physical activity. Motivational signage has been successful in promoting stair usage in various settings. This study was to investigate the effects of motivational signage on stair usage in a Hispanic serving institution. Participants: A total of 31,067 pedestrians were observed from February to March 2013. Methods: Stair usage was monitored for 9 h per day each week at phase 1 (baseline), 2 (intervention), and 3 (post-intervention). Results: Overall, participants' stair usage was higher during phase 2 (49.0%) and phase 3 (48.0%), compared with phase 1 (39.7%). The participants during phase 2 and 3 were more likely to use the stairs compared to participants during phase 1, regardless of floor level (3-story or 4-story building), status (student or staff/faculty), and time of day (7:30-10:29, 10:30-13:29, or 13:30-16:30) (p < 0.001). Conclusion: Motivational signage can effectively encourage more stair usage, and hence promote healthy behavior in a predominantly Hispanic-serving institution.