Introduction: The six-month course of isoniazid preventive therapy (IPT) has been demonstrated to have significant benefits in mitigating the occurrence of tuberculosis (TB), particularly in high TB burden settings. However, its implementation in Sub-Saharan countries remains subdued for obscure reasons. This study investigated the factors inhibiting IPT uptake in the sub-Saharan country of Lesotho, which has one of the highest rates of TB incidences globally. Material and methods: Data were obtained from 46 healthcare workers, key informants at the Ministry of Health of Lesotho, and representatives of partner organizations, who were purposively selected for their roles in IPT implementation. Data were coded to identify themes, and the emerging themes were benchmarked to previous typologies for evaluating the implementation of best practices in health interventions, namely effectiveness, reach, sustainability, and adaptation. Each major theme was further linked to the World Health Organization's 'six building blocks of a health system'. Results: Challenges affecting the implementation of IPT were as follows: ineffective TB screening due to challenged decentralization of human immunodeficiency virus (HIV)/TB services, late detection of side effects linked to weak monitoring systems, and inefficient health information systems. Further challenges in the health system included interrupted supply chains due to insufficient health system financing, while inadequate healthcare workers' education on IPT implementation was also noted. Conclusions: These findings indicate that a wide spectrum of challenges has affected the implementation of IPT in Lesotho, and this indicates the need for 'health systems approach' to the implementation of IPT in Lesotho and other countries with similar challenges.