Background: Tuberculosis accounts for almost half a million human immunodeficiency virus (HIV) associated deaths. Provision of isoniazid preventive therapy (IPT) is one of the public health interventions for the prevention of TB in HIV infected individuals. However in Tanzania the data on proportion of acceptance and associated factors for IPT uptake is limited. Therefore, the current study was aimed to determine the acceptability, defaulter and factors associated with IPT uptake among PLHIV who attended CTC at KCMC from January 2015 to January 2016. Methodology: The study was a retrospective cross sectional study using existing data from medical record. The study included all HIV positive patients who attended CTC at KCMC from January 2015 to January 2016. HIV positive patients who were below 15 years and those with confirmed TB infection at the time of enrollment to IPT were excluded from the study. Data was collected by researcher using data extraction sheet. Results: A total of 183 clients were enrolled in this study, of which 121 (66.1%) were females, 99 (54.1%) were in age group 30-49 years, 142 (77.6%) were Christians and nearly half 90 (49.2%) were unemployed. 180 (98.4%) accepted IPT. Seven (3.9%) defaulted from IPT. Percentage of defaulting was greater in males (6.5%) than in females (2.5%). Unemployed and those with WHO clinical stage two and three of HIV seemed to have higher defaulting proportion than their counterparts. However these were not statistically significant. During the course of follow up there was development of active TB in 1(0.6%) participant and Isoniazid side effect in 1(0.6%) participant. Conclusion: IPT acceptance and the completion of its treatment course are high. This means that HIV infected patients are ready to accept and complete IPT. The proportion of acceptance was more in males than in females. IPT defaulter in this study is very low, suggesting that most of the HIV positive patients attending CTC at KCMC completed the required dose of IPT. Therefore, TB incidence can be reduced in PLHIV. Proportion of defaulter was more in males than in females, therefore special intervention need to be invented to boost the completion of IPT among males.