Objective Agreeable individuals report more intense withdrawal‐oriented negative emotions across aversive situations. Two studies tested the hypothesis that self‐regulatory depletion (i.e., ego depletion) moderates the relationship between trait Agreeableness and negative emotional responding. Method Ego depletion was manipulated using a writing task. Emotional responding was measured with startle eye‐blink responses (Study 1, N = 71) and self‐reported valence, arousal, and empathic concern (Study 2, N = 256) during emotional picture viewing. Trait Agreeableness was measured using a questionnaire. Results In Study 1, Agreeableness predicted especially large startle responses during aversive images and especially small startles during appetitive images. After exercising self‐control, the relationship between startle magnitudes and Agreeableness decreased. In Study 2, Agreeableness predicted more empathic concern for aversive images, which in turn predicted heightened self‐reported negative emotions. After exercising self‐control, the relationship between Agreeableness and empathic concern decreased. Conclusions Agreeable individuals exhibit heightened negative emotional responding. Ego depletion reduced the link between Agreeableness and negative emotional responding in Study 1 and moderated the indirect effect of Agreeableness on negative emotional responding via empathic concern in Study 2. Empathic concern appears to be a resource‐intensive process underlying heightened responding to aversive stimuli among agreeable persons.