Poor adherence to medication can lead to treatment failure in healthcare workers (HWCs) with latent tuberculosis infection (LTBI) who are at high risk of developing active tuberculosis. However, the factors associated with non-completion of nine-month LTBI treatment with isoniazid (9 H) have not been well studied. We investigated the completion rate and factors affecting adherence to LTBI treatment with 9 H among HCWs. A prospective cohort study of 114 HCWs who were diagnosed with LTBI by QuantiFERON-TB Gold In-Tube tests were performed in a single university hospital between June 2016 and December 2017. All patients received the 9 H LTBI treatment. At each visit, treatment adherence and development of adverse reactions to isoniazid were evaluated via a standard questionnaire. To evaluate the impact of the severity of hepatotoxicity on non-completion of LTBI treatment, we classified hepatotoxicity into two groups: severe hepatotoxicity was defined as alanine aminotransferase >3.0 times the upper normal limit (UNL) with symptoms or = 5.0 times the UNL. Mild hepatotoxicity was defined as alanine aminotransferase>UNL, but not meet the definition of severe hepatotoxicity. Overall, 71 HCWs (62.3%) completed LTBI treatment with 9 H while 43 HCWs (37.7%) discontinued their treatment. Most discontinuation (81.4%, 35/43) occurred during the first three months of treatment. There were no significant differences in age, sex, occupation, or comorbidities between the HCWs who completed and those who discontinued LTBI treatment. However, HCWs who discontinued LTBI treatment had more hepatotoxicity than those who completed treatment (44.2% vs. 11.3%, P < 0.001). Cox proportional hazard analysis revealed that hepatotoxicity is the only factor significantly associated with discontinuation of 9 H LTBI treatment (unadjusted HR = 2.89, 95% CI = 1.62–5.46). In multivariable analysis, not only severe hepatotoxicity (adjusted HR = 7.99, 95% CI = 3.05–20.94) but also mild hepatotoxicity was significantly associated with discontinuation of LTBI treatment (adjusted HR = 2.34, 95% CI = 1.05–5.21). The completion rate of 9 H LTBI treatment was 62.3% among HCWs. While age, sex, occupation, and pretreatment comorbidities were not associated with treatment completion, isoniazid-induced hepatotoxicity significantly affected adherence.