Nicotine is an alkaloid that is used by millions of people around the world through smoking and Effects on the body, especially the endocrine system. On the other hand, millions of people in the world suffer from diabetes. Hence, this study investigated the effects of nicotine on serum levels of the hormone glucagon and glucose was performed in adult male rats. This study is an experimental study. In this research, 50 male mature rats with an average weight of 220-250 grams were enrolled as the experiment, witness and control groups. The experimental group was divided into 3 subgroups and 0.5, 1 and 1.5 mg per kg of nicotine was injected intraperitoneally for 5 days. Around the same time every day 1 ml Physiological serum was given to the witness group, but the control group did not receive anything. At the end of 6 days, blood sample was taken from the heart ventral areas and the Serum levels of glucagon hormone were measured using radioimmunoassay (RIA) method and the commercial kits. The results were evaluated using one-way ANOVA. According to the results of this study, Nicotine at all doses significantly increased serum levels of the hormone glucagon and glucose and the dosage increased, the rate of increase is higher (P<0.01). The results of this study can be concluded that nicotine which stimulate glucagon secretion.