Mitochondrial DNA 5178 adenine/cytosine (mt5178 A/C) polymorphism is one of the longevity-associated mitochondrial DNA polymorphisms. The frequency of the mt5178A genotype is significantly higher in Japanese centenarians than in the general population. We previously reported that serum high-density lipoprotein cholesterol levels were significantly higher in men with mt5178A than in those with mt5178C. However, this significant difference disappeared after adjusting for drinking frequency. To investigate the interaction between mt5178 A/C polymorphism and habitual drinking on serum lipid levels, we performed an association study in 321 healthy middle-aged Japanese men. Interaction between mt5178 A/C polymorphism and daily drinking on serum triglyceride (TG) levels was observed (P=0.019). Moreover, interaction between mt5178 A/C polymorphism and cigarette consumption on serum TG levels was also observed (P=0.022). Multiple regression analysis showed that, in men with mt5178A, daily drinking decreased TG levels (P=0.025), and cigarette consumption increased TG levels (P<0.001), while in men with mt5178C, the effects of daily drinking and cigarette consumption on TG levels were unclear. No interaction was observed on other lipid levels. Longevity-associated mitochondrial DNA 5178 A/C polymorphism thus influences the effects of daily drinking and cigarette consumption on TG levels in middle-aged Japanese men.