Objective: Botulinum toxin (BoTX) injection to the longus colli (LCo) muscle has been demonstrated to have a role in treating cervical dystonic (CD) patients. It can, however, cause critical complications and awareness of such complications is required. Currently, there is no substantial information regarding this novel procedure. This study aims to define the potentially safe method of injection based on assessment of anatomical measurements.Materials and Methods: We examined distances between the puncture sites and adjacent structures in Thiel-embalmed human cadavers (n=20) to propose an alternative technique for BoTX injection. Parameters were examined for the medial and lateral approaches at the fifth and sixth cervical vertebral levels. We compared each variable between the two different vertebral levels and the two different approaches to evaluate statistical differences.Results: Comparing distances between the puncture sites and neck anatomical structures in each of the two approaches, results were statistically significant. Similarly, we found using the medial approach statistical significance when comparing the measurements at the fifth with the sixth cervical vertebral level of the distances between the puncture sites and the thyroidal arteries and recurrent laryngeal nerve (p < 0.05).Conclusion: The present study results provide initial guidelines for the safe technique for BoTX injection into LCo. Our findings suggest that the medial approach at the C6 vertebral level is preferable with minimal injury to vital structures. Thus, it may provide an optional method and can be used as guidance to improve surgical practice. Ethical approval was not required for this study.