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. 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. 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. 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. 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.