Mice have gained more and more attention in recent years and been widely used in transgenic experiments. Although the number of researches on the heart rate variability (HRV) of mice has been gradually increasing, a consensus on the frequency ranges of autonomic modulation has not been established. Therefore, the main purpose of this study was to find a HRV “prototype” for conscious mice in the state of being motionless and breathing regularly (called “genuinely resting”), and to determine the frequency ranges corresponding to the autonomic modulation. Further, whether these frequencies will change when the mice move freely was studied to evaluate the feasibility of the HRV spectrum as an index of the autonomic modulation of mice. The recording sites were specially arranged to simultaneously obtain the electrocardiography and electromyography data to be provided for the use of HRV analysis and motion monitoring, respectively. The states of being motionless and breathing regularly as judged from the electromyography results were selected as a genuine resting state of a conscious mouse. The frequencies related to autonomic modulation of HRV were determined by comparing the spectrum changes before and after blockades of the autonomic tone by different pharmaceutical agents in both the genuine resting state and freely moving states. Our results showed that the HRV of mice is not suitable for indexing sympathetic modulation; however, it is possible to use the spectral power in the frequency range between 0.1 and 1 Hz as an index of parasympathetic modulation.