Background: Despite evidence that isoniazid preventive therapy (IPT) can reduce the risk of tuberculosis (TB) disease among People Living with Human Immunodeficiency Virus (PLHIV), uptake of IPT is low in many resource-limited settings. This study determined the level of IPT uptake and its associated factors amongst PLHIV. Materials and Methods: This was a retrospective quantitative study amongst PLHIV who do not have active TB and enrolled in 2019 - 2020 for anti-retroviral therapy (ART) in Butebo district, Uganda. Data related to demographic factors (age, sex, religion, marital status, employment status, education level, area of residence, household density), health facility factors (pre-IPT counseling), community factors [distance from Health Center (H/C), incurred costs to reach H/C], and IPT drug-related factors [frequency of Isoniazid (INH) refill, INH stock-outs] were collected from four health facilities using a checklist. The data was analyzed into descriptive statistics and relationships determined using Chi-square tests. Results: Among eligible PLHIV (272), 34.2% achieved IPT uptake. The mean duration between HIV diagnosis and the start of IPT was 4.31 years, with IPT Uptake among males (37.0%), females (32.8%), married (39.5%), and Christians (35.4%). Factors that affect the rate of IPT uptake include employment, education, residence, costs to reach H/C, and preIPT counseling. The IPT completion rate was 97.8%. All the cases who had regular INH refill completed IPT compared to 60.0% with the irregular refill, while 97.8% did not experience INH stock-outs and completed IPT. Conclusion Pre-IPT counseling was the most significant contributing factor for IPT uptake.IPT uptake may be scaled up by integrating IPT services in routine HIV care, enhancing counseling for IPT and supervision and monitoring, training of health workers, and improving logistical supplies at the health centers.