Background: Tuberculosis treatment interruption is a failure of attending two scheduled appointments to collect the drugs in either phase of tuberculosis treatment. Even if TB treatment is crucial to achieve a cure and avoid the emergence of drug resistance, treatment interruption is the most testing and deterring factor for successful tuberculosis treatment and one of the problems leading to the development of drug-resistant tuberculosis. TB treatment interruption is the precursor for loss to follow-up and treatment failure, but the magnitude of this problem is unknown in Ethiopia. Thus, this study was intended to identify determinants of treatment interruption among drug-susceptible pulmonary tuberculosis patients in South Ari district, Southern Ethiopia. Methods: An institution-based unmatched case control study was conducted from February through April 2020 using 255 samples with a ratio of 2:1 (controls to cases). Data were entered into Epi data version 4.2 and exported for analysis using STATA 14.0 statistical software. The variables having a p-value of less than 0.25 in the bivariable analysis were subjected to multivariable logistic regression analysis. In multivariable logistic regression analysis, AORs, 95% CIs, and p-values of <0.05 were used to identify significant variables. Results: The median age was 34 (IQR: 18) years in cases and 29 (IQR: 16) years in control groups. Significant factors that were associated with treatment interruption were alcohol consumption (AOR = 2.99, 95% CI; 1.41-6.36); smoking habits (AOR = 2.82, 95% CI; 1.14-6.94); use of traditional medicine (AOR = 2.35, 95% CI 1.05-5.24); co-infected with HIV (AOR=1.58, 95% CI; 1.85-4.29), and waiting time at the health facility ≥30 minutes (AOR = 2.98, 95% CI; 1.31-6.80). Conclusion: Alcohol consumption, waiting time at the health facility ≥30 minutes, smoking habits, used traditional medicine, and HIV co-infected were potential determinants. Enhancing public health education, designing strategies that emphasize patients with HIV coinfection, and reducing waiting times are recommended.