The separation and localization of sound source signals are important techniques for many applications, such as highly realistic communication and speech recognition systems. These systems are expected to work without such prior information as the number of sound sources and the environmental conditions. In this paper, we developed a dodecahedral microphone array and proposed a novel separation method with our developed device. This method refers to human sound localization cues and uses acoustical characteristics obtained by the shape of the dodecahedral microphone array. Moreover, this method includes an estimation method of the number of sound sources that can operate without prior information. The sound source separation performances were evaluated under simulated and actual reverberant conditions, and the results were compared with the conventional method. The experimental results showed that our separation performance outperformed the conventional method.