The present study sought to establish whether increases in negative affect following exposure to homosexuality mediate the relation between sexual prejudice and anger network activation. Participants were 159 heterosexual men who completed measures of sexual prejudice and state negative affect. Participants were randomly assigned to view a short video depicting either a male-male or male-female sexual interaction and, following the video, again reported state negative affect. Anger network activation was then assessed via response latencies from a lexical decision task that required participants to identify emotion or emotionally neutral words from non-words. Results indicated that the pattern of covariation between sexual prejudice and anger facilitation was significantly more positive when participants viewed male-male erotica relative to male-female erotica. Increases in negative affect, particularly anxiety/fear, were found to mediate this association. These findings suggest that increases in negative affect following exposure to homosexuality may be the emotional trigger for biases in the processing of anger-related information and represent the cognitive underpinnings of sexually prejudiced aggression toward gay men.