The first certified reference cigarette, 1R6F, was produced by the Center for Tobacco Reference Products at the University of Kentucky in 2015 and certified in 2016. 1R6F reference cigarettes have been stored at -20 °C since they were manufactured. 1R6F has been widely used as a control cigarette or a monitor for nonclinical investigational purposes in tobacco product analysis and scientific research. However, there is little published data to demonstrate the stability of the 1R6F cigarette. In this paper, we report the results of a long-term storage study of the 1R6F cigarette tobacco filler and the resulting mainstream smoke. 1R6F cigarettes were stored under different conditions (room temperature, refrigerator (4 °C), and freezer (-20 °C)) for 3 years since April 2017. The constituents in the cigarette tobacco filler (oven volatiles, nicotine, N'-nitrosornicotine (NNN), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)) and the mainstream smoke (nicotine, NNN, NNK, benzo[α]pyrene, carbon monoxide, total particulate matter) were analyzed. Some physical parameters (resistance to draw and ventilation) were also measured. Analysis of our data showed that no significant differences in these major constituents were detected after storage of the 1R6F cigarette at -20 °C for 3 years.