Background: In recent years, childhood tuberculosis (TB) has attracted global interest after a long period of neglect. We aimed to give an update about the epidemiological profile of childhood TB in south of Tunisia and to describe their chronological trends from 1995 to 2016. Methods: We retrospectively collected data of TB new cases in children aged <15 years between 1995 and 2016 in Southern Tunisia. Joinpoint Regression Analysis was performed to analyze chronological trends and annual percentage changes (APC) were estimated. Results: Overall, 204 cases of TB were noted in children. The average incidence rate of overall TB was 4.09/100000 population/year. There was a significant rise in extrapulmonary tuberculosis (EPTB) incidence (APC=2.76%; 95% confidence interval (95% CI)=[0.40-5.00]), while pulmonary tuberculosis PTB incidence rate showed a non-significant decrease over time. The under-fives had a significant growing trend (APC of 3.95%; 95% CI=[0.80-7.30]). We noted a significant upward trend of TB incidence in rural districts (APC=4.91%; 95% CI=[1.90-8.10]). Trends analysis by anatomical site indicated an increase in lymph node TB new cases (APC=5.26%; 95% CI=[2.80-7.80]). Conclusions: A significant rise in TB incidence rate was observed in TB cases, EPTB, notably lymph node, the under-fives and rural districts inhabitants. An increased focus on groups at high risk and implementing preventive strategies in this highly vulnerable population is warranted.