We examined puberty-specific effects on affect-related behavior and on the psychophysiology of defensive and appetitive motivation while controlling for age. Adolescents (N = 94, ages = 12 and 13 years) viewed 75 pictures (International Affective Picture System: pleasant, neutral, and aversive) while listening to auditory probes. Startle response and postauricular (PA) reflex were collected as measures of defensive and appetitive motivation, respectively. Pubertal status and measures of anxiety/stress reaction and sensation/thrill seeking were obtained. Mid-/late pubertal adolescents showed enhanced startle amplitude across all picture valences. A Puberty × Valence interaction revealed that mid-/late pubertal adolescents showed appetitive potentiation of the PA, whereas pre-/early pubertal adolescents showed no modulation of the PA reflex. Mid-/late pubertal adolescents also scored significantly higher on measures of sensation/thrill seeking than did their pre-/early pubertal peers and puberty moderated the association between psychophysiology and behavioral measures, suggesting that it plays a role in reorganizing defensive and appetitive motivational systems.