Electronic cigarettes (e-cigarettes) have been marketed as a less lethal substitute for smoking traditional cigarettes. This study aims to investigate the impact of e-cigarettes aerosol exposure on lactating dams and pups, whose dams were exposed. Lactating dams received fresh air (control) or e-cigarettes aerosol during lactation (day 4–21). Maternal exposure to e-cigarettes aerosol during lactation induced significant reduction (P < 0.0001) in the fat content of the milk and serum Leptin level (P < 0.005) compared to control dams. Furthermore, pups whose dams were exposed to e-cigarettes during lactation showed an increased level of glucose, thyroxine and decreased level of insulin. The exposure to e-cigarettes aerosol during lactation altered the composition of milk as well as the hormonal and biochemical profile in dams and pups. This result, if observed in women using e-cigarettes, suggests that e-cigarettes’ use during lactation may have consequences on the milk production and hormonal and biochemical profile in breastfeeding mothers and nursing babies.