Yamamoto and Ikegami (2019) hypothesized that third parties infer the helper’s motive based on the naive self-presentation theory and demonstrated the effect of the observers’ presence on motive inference. This study aimed to replicate this effect and provide convincing evidence for this hypothesis by manipulating the observers’ gender. We conducted a questionnaire experiment with 277 undergraduates using scenarios of helping experiences. Similar to Yamamoto and Ikegami (2019), the results showed that self-presentational motives were more likely to be attributed to helpers in the presence versus absence of observers. Importantly, the results showed that self-presentational motives were more likely to be attributed to helpers when there was an opposite gender versus a same-gender observer. However, there was no difference in motive inference between the conditions when there was a mere opposite-gender observer versus an opposite-gender observer with whom the helper was in love. The effects of the observers’ presence and their gender on motive inference were consistently confirmed across the two types of helping scenes. This study successfully provided convincing evidence for the above hypothesis.