Background: The issue of lower extremity amputation has been in the Colombian political agenda for its relationship with the armed conflict and antipersonnel mines. In 2015 the Colombian Ministry of Health published a national clinical practice guideline (CPG) for amputee patients. However, there is a need to design implementation strategies that target end-users and the context in which the CPG will be used. This study aims to identify users' perceptions about the barriers and facilitators for implementing the guideline for the care of amputee patients in a middle-income country such as Colombia. Methods: Semi-structured interviews were conducted with 38 users, including patients, health workers, and administrative staff of institutions of the health system in Colombia. Individuals were purposively selected to ensure different perspectives, allowing a balance of individual positions. Results: According to participants' perceptions, barriers to implementation are classified as individual barriers (characteristics of the amputee patient and professionals), health system barriers (resource availability, timely care, information systems, service costs, and regulatory changes), and barriers related to clinical practice guidelines (utility, methodological rigour, implementation flexibility, and characteristics of the group developing the guidelines). Conclusions: Our study advances knowledge on the perceived individual and health system barriers and facilitators for the implementation of the CPG for amputee patients in Colombia. Importantly, the governance, financial, and service delivery arrangements of the Colombian health system are determining factors in implementing CPGs. For example, the financial arrangements between the insurance companies and the health care provider institutions were identified as barriers for the implementation of recommendations related to the continuity and opportunity of care of patients with amputations. The design of implementation strategies that successfully address the individual behaviours and the contextual health systems arrangements may significantly impact the health care process for amputee patients in Colombia.