Background Minimally invasive surgery in skull base relying on searching for possible anatomic basis for endoscopic technology is controversial. The objective of this study was to observe the spatial relationships between main blood vessels and nerves in the cerebellopontine angle area and provide anatomic basis for lateral and posterior skull base minimally invasive surgery via endoscopic retrosigmoid keyhole approach. Methods This study was conducted on thirty dried adult skulls to measure the spatial relationships among the surface bony marks of posterior cranial fossa, and to locate the most appropriate drilling area for retrosigmoid keyhole approach. In addition, we used 10 formaldehyde-fixed adult cadaver specimens for simulating endoscopic retrosigmoid approach to determine the visible scope. Results The midpoint between the mastoid tip and the asterion was the best drilling point for retrosigmoid approach. A hole centered on this point with the 2.0 cm in diameter was suitable for exposing the related structures in the cerebellopontine angle. Retrosigmoid keyhole approach can decrease the pressure on the cerebellum and expose the related structures effectively which include facial nerve, vestibulocochlear nerve, trigerninal nerve, glossopharyngeal nerve, vagus nerve, accessory nerve, hypoglossal nerve, anterior inferior cerebellar artery, posterior inferior cerebellar artery and labyrinthine artery, etc. Conclusions Exact location on endoscope retrosigmoid approach can avoid dragging cerebellum during the minimally invasive surgery. The application of retrosigmoid keyhole approach will extend the application of endoscopic technology.