Objectives: To investigate whether the introduction of tobacco plain packaging in Australia from 1 October 2012 was associated with a change in the number of calls to the smoking cessation helpline, Quitline, and to compare this with the impact of the introduction of graphic health warnings from 1 March 2006. Design and setting: Whole-of-population interrupted time-series analysis in New South Wales and the Australian Capital Territory between 1 March 2005 and October 2006 for the comparator, graphic health warnings, and October 2011 and April 2013 for the intervention of interest, tobacco plain packaging. Main outcome measure: Weekly number of calls to the Quitline, after adjusting for seasonal trends, anti-tobacco advertising, cigarette costliness and the number of smokers in the community. Results: There was a 78% increase in the number of calls to the Quitline associated with the introduction of plain packaging (baseline, 363/week; peak, 651/week [95% CI, 523-780/week; P < 0.001]). This peak occurred 4 weeks after the initial appearance of plain packaging and has been prolonged. The 2006 introduction of graphic health warnings had the same relative increase in calls (84%; baseline, 910/week; peak, 1673/week [95% CI, 1383-1963/week; P < 0.001]) but the impact of plain packaging has continued for longer. Conclusions: There has been a sustained increase in calls to the Quitline after the introduction of tobacco plain packaging. This increase is not attributable to anti-tobacco advertising activity, cigarette price increases nor other identifiable causes. This is an important incremental step in comprehensive tobacco control.