Background: Tuberculosis (TB) is the most frequent life-threatening infection and a common cause of death for people living with HIV (PLHIV). The influence of TB and HIV infection has enhanced the magnitude of both epidemics. Several clinical interventions recommended early diagnosis in PLHIV and treating latent TB infection (LTBI) with Isoniazid preventive therapy (IPT) along with antiretroviral therapy (ART). IPT is one of the key interventions recommended by the world health organization (WHO) for the prevention of TB in patients infected with HIV. Hence, this study aimed to determine IPT utilization rate among adult HIV infected patients enrolled in HIV care and qualitative analysis, which explore the factors that influence IPT use among PLHIV under follow-up, Health care providers (HCPs) and TB/HIV coordinators working in Jimma University Specialized Hospital (JUSH) ART clinic. Methods: An Institution based mixed cross-sectional study was conducted in JUSH ART clinic. Adult HIV infected patients were enrolled by a systematic sampling technique from the registered medical records of JUSH HIV care. PLHIV who were on follow-up and eligible for IPT during the study period, permanent HCPs and TB/HIV coordinators working in ART clinic were included in the qualitative investigation using semi-structured questioners and in-depth interviews. All statistical analysis was compiled by Epi data 3.1 and SPSS 20. Results: Demographic and clinical factors are not significantly associated with IPT use but ethnicity (P≤ 0.02**) was highly significant with IPT use in logistic regression model. Overall, 59.2% of the patients have been prescribed and taken at least one-month course of IPT. The results of in-depth interviews are grouped into three core categories as patient perceptions, HCPs and TB/HIV coordinator perspectives. Discussion and conclusion: PLHIV, HCPs and TB/HIV coordinators suggested their overall response as periodic counseling for target groups, educating the benefits of IPT and increasing public awareness on TB prophylaxis in PLHIV will increase the acceptance and implementation of IPT in large scale.Higher attention should be provided in linking all HIV patients to the nearest health facilities for receiving free service packages and medical care.