The effects of the motivational states of public speakers, such as rejection avoidance needs, as well as the presence or absence of an audience on psychophysiological responses when expecting to make a speech were investigated. Participants delivered a three-minute speech in a room with or without an audience. Participants completed the General Affects Scales before and after the speech, and the negative affect (NA) subscale scores of the scale were used to assess their psychological state. Salivary cortisol level was measured before and after the speech as an index of their physiological response. The results indicated that speakers with high rejection avoidance needs had greater NA prior to the speech regardless of the presence or absence of an audience. Moreover, they had increased cortisol levels only when speaking in front of an audience. Speakers with low rejection avoidance needs had greater NA when speaking in front of an audience, whereas they did not show increased cortisol levels, regardless of the presence or absence of an audience. These results suggest that when a speaker expects to deliver a speech, the speaker's motivational state and the presence or absence of an audience interactively cause changes in psychophysiological responses.