Melatonin has been suggested to affect human emotion, but conflicting evidence exists. Therefore, we tested the effect of a single dose of a 4 mg prolonged release formulation of melatonin on a biologically based model of emotional processing. Affective modulation of acoustic white noise startle (103 dB) by emotional slides selected from the International Affective Picture System (IAPS) was assessed in 16 healthy young women twice, in a double-blind, placebo-controlled, balanced cross-over design. Melatonin significantly reduced startle responsiveness, but did not impact affective startle modulation, nor startle habituation. Melatonin significantly reduced arousal ratings and induced a parasympathetically dominated heart rate variability pattern indicative of a non-aroused state. We conclude that melatonin reduces arousal and startle responsiveness. However, no evidence for a direct emotion-modulating effect of melatonin was found in this healthy cohort.