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Background Our goal was to identify brain structures responsible for pain-related autonomic changes by the correlation of simultaneously acquired functional magnetic resonance imaging (fMRI) and electrocardiogram (ECG) data. Methods Eighteen healthy men (age: 22.89 ± 1.96) were involved. Painful sensation was evoked by heat. Simultaneously recorded brain fMRI and ECG data during pain were compared to data acquired during a non-painful heat sensation. From the ECG data, time- and frequency domain parameters of heart rate variability (HRV) were extracted. Results We found that: (1) among the common elements of both pain network and central autonomic network (CAN) only the medial prefrontal frontal cortex (MPFC) showed significant correlation with HRV; (2) the parasympathetic response to the painful stimuli showed a positive, while the sympathetic response a negative association with pain related BOLD-signal change observed in MPFC; (3) time domain parameters of HRV were negatively associated with MPFC activation. Conclusions The novelty of our study—compared to previous ECG–fMRI studies—is that we used pain as stimulus and investigated both frequency- and time-domain parameters of HRV. Compared to other stimuli used in earlier studies to activate the CAN, pain sensation can be standardized easier and might allow us to better understand the functional organization of CAN. The results of the current ECG–fMRI study may have direct clinical relevance in understanding the pathomechanisms of several clinical conditions. Perspective There are some simultaneous ECG–fMRI and ECG–Positron Emission Tomography (PET) studies, but limited information is available about the pain-related brain function–HRV relations. The novelty of our study is that we used pain as stimulus to activate the central autonomic network and investigated both frequency- and time-domain parameters of HRV.