Evidence suggests that suboccipital musculature plays an important role in headache. Proper therapeutic approaches targeting this muscle are needed. Our aim was to determine with fresh cadavers and ultrasound imaging if a solid needle is able to properly penetrate the obliquus capitis inferior muscle during the application of dry needling. A cadaveric and human descriptive study. Needling insertion and ultrasound imaging of the obliquus capitis inferior muscle was conducted on 10 pain-free healthy subjects and 5 fresh cadavers. Needling insertion was performed using a 40 mm needle inserted midway between the spinous process of C2 and transverse process of C1. The needle was advanced from a posterior to anterior direction into the obliquus capitis inferior muscle with an inferior-lateral angle to reach the lamina of C2. For the cadaveric study, the obliquus capitis inferior placement was verified by resecting the superficial upper trapezius, splenius capitis, and semispinalis capitis muscles. For ultrasographic study, a linear transducer was aligned with the long axis of the obliquus capitis inferior muscle after needle insertion. Both the cadaveric and ultrasonic studies showed that the needle penetrated the obliquus capitis inferior muscle during its insertion and that the tip of the needle rested against C2 laminae, thereby reaching the targeted muscle. This anatomical and ultrasound imaging study supports the assertion that needling insertion of the obliquus capitis inferior muscle can be properly conducted by an experienced clinician.