Primate ecotourism is a fast-growing tourism sector that may have a negative effect on wildlife. In riparian areas, tourists can conveniently reach primates via motor boats, but no study has directly examined whether such boats cause stress in primates. Our goal was to test whether the approach of a motor boat induces stress-related and other behaviors in proboscis monkeys (Nasalis larvatus), an Endangered species. We studied six one-male, multifemale groups living in a remote riparian area in Sabah, Malaysia, and conducted an experiment by approaching the monkeys in a motor boat by using three conditions with different speeds and travel distances (fast-close, slow-close, and slow-far conditions; 7-8 subjects per condition). For each condition, we compared stress-related behaviors before the boat approach with after the boat started approaching. Feeding, allogrooming and aggression were similarly examined, respectively. We also observed the monkeys’ behaviors at other times to examine age-sex classes differences in vigilance, social proximity, allogrooming, aggression and play (87 subjects). In the experiment, subjects displayed stress-related behaviors for longer in the fast-close and slow-close conditions once the boat started approaching than before the boat approach. The subjects also reduced feeding in the fast-close condition after the boat started approaching. In our observational study, males were more vigilant than females—a behavior that is likely to relate to male-male competition and group protection. This study provides evidence that even a single motor boat moving slowly, with humans behaving calmly, may negatively affect primate behavior and induce stress—an impact that is likely to be larger with tourist boats. Our study also shows that using conditions comparable to the slow-far condition (speed of 3.6 km/hr; no closer than 60 m), where no impact was observed, may help with to develop guidelines for primate tourism in riparian areas. Future research that examines the impact of boats on other primates is needed.