Although possible sources and functions of the resting-state networks (RSNs) of the brain have been proposed, most evidence relies on circular logic and reverse inference. We propose that autonomic arousal provides an objective index of psychophysiological states during rest that may also function as a driving source of the activity and connectivity of RSNs. Recording blood oxygenation level-dependent (BOLD) signal using functional magnetic resonance imaging and skin conductance simultaneously during rest in human subjects, we found that the spontaneous fluctuations of BOLD signals in key nodes of RSNs are associated with changes in nonspecific skin conductance response, a sensitive psychophysiological index of autonomic arousal. Our findings provide evidence of an important role for the autonomic nervous system to the spontaneous activity of the brain during “rest.”