This study investigates changes of stress-related psychophysiological reactions after treatment with EMDR. Sixteen patients with posttraumatic stress disorder (PTSD) following type I trauma underwent psychometric and psychophysiological assessment during exposure to script-driven imagery before and after EMDR and at 6-month follow-up. Psychophysiological assessment included heart rate (HR) and heart rate variability (HRV) during a neutral task and during trauma script listening. PTSD symptoms as assessed by questionnaire decreased significantly after treatment and during follow-up in comparison to pretreatment. After EMDR, stress-related HR reactions during trauma script were significantly reduced, while HRV indicating parasympathetic tone increased both during neutral script and during trauma script. These results were maintained during the follow-up assessment. Successful EMDR treatment may be associated with reduced psychophysiological stress reactions and heightened parasympathetic tone.