Background: Despite the implementation of Directly Observed Treatment Short-course (DOTS) strategy in all public health centers in Papua Province, Indonesia, since 1998, the rate of loss to follow-up (LTFU) during tuberculosis (TB) treatment remains high (above 16%). Objectives: We aimed to identify factors associated with non-adherence during TB treatment among patients treated at public health centers (PHCs) in Jayapura, Papua. Method: We conducted a case-control study including new TB patients registered at eight PHCs from 2007 to 2009. Non-adherent cases were TB patients with a history of not taking anti-TB drugs for >2 consecutive weeks or >30 days cumulatively. Controls were randomly selected from patients who completed all doses of TB drugs in time. Data were collected by face-to-face interview using a pre-structured questionnaire and analyzed with logistic regression models. Results: Data were available for 81 of 103 eligible cases and 183 of 206 eligible controls. Difficult access to healthcare (i.e. reported to have a problem with distance/travel cost and history of moving residence in the past year), lack of TB knowledge (i.e. lack of knowledge about TB transmission and the cause of TB and unawareness of the consequences of stopping TB treatment), and treatment experience (i.e. lack of TB education provided by TB nurse and the use of loose vs. fixed-dose combinations) were associated with non-adherence during TB treatment in the adjusted model, as were being aged under 35 years and having a history of TB in the family. Conclusion: Our results suggest the need to improve TB treatment delivery especially to those who have difficult access to healthcare, and to routinely provide education to increase patients’ knowledge about TB and TB treatment. In addition, more attention to younger patients and those with a history of TB in their family is also needed.