Trade in lion parts associated with cultural and traditional use is poorly understood. Here we sought to better understand the role and use of lion body parts in the commercial traditional medicine (muthi) trade of South Africa. In 2019 we conducted a semi-structured questionnaire survey of muthi traders (n = 10) and traditional healers (n = 20) which explored the significance and symbolism of lions, traded parts and preferences, sources and supply of lion parts, and perceived sustainability of lion derivatives in the South African muthi trade. Our results suggest a cultural importance of lion associated with the umndawu ancestral spirit in particular, as well as in the training and practice of cultural-spiritual healers. Lion paws and parts thereof were most frequently reported as sold by traders and demanded by healers, correlating with recent trends in body-part removals from lion mortalities. Respondents indicated that lion parts were obtained from a variety of sources including wild lion populations in neighbouring countries and captive-breeding farms. Our findings are discussed relative to current concerns in lion conservation and highlight a need for further understanding of the traditional medicine complex, the influence that ancestral spirits have on lion body-part trade, and increased engagement with traditional medicine stakeholders.