Objective: Previous research has demonstrated that prefrontal activity is related to control over stress responses. However, the causal mechanisms are not well understood. In this study we investigated the possible influence of brain stimulation on the physiological stress response system. Because an increased stress response is known to precipitate psychiatric disorders, further inquiry can have important clinical implications. Method: In 38 healthy, right-handed female participants, we examined the effects of a single sham-controlled high-frequency (HF) repetitive Transcranial Magnetic Stimulation (rTMS) session over the left (n = 19) and right (n = 19) dorsolateral prefrontal cortex (DLPFC) on the autonomic nervous system stress response, as measured by heart rate variability (HRV). Stress was transiently induced through evaluative negative feedbacks. Results: Although the induction procedure was efficient in increasing self-reported distress in all groups and conditions, only after real HF-rTMS over the left DLPFC the physiological stress response was diminished, as indicated by a significant increase in HRV. No effects were found in the sham or right side stimulation condition. Conclusions: These findings demonstrate that increasing brain activity by HF-rTMS over the left DLPFC can help attenuating physiological stress reactions. Results are indicative of the positive effects of rTMS on stress resilience and underscore the possible benefit of HF-rTMS as a transdiagnostic intervention. Finally, the results also show that effects only occur when stimulating the left DLPFC, which is in line with the therapeutic effects of HF-rTMS in affective disorders.