A sense of humor is widely viewed as beneficial for physical health. However, some limited research suggests that humor may actually be related to increased smoking and alcohol consumption because humorous individuals may take a less serious attitude toward substance use. The purpose of the present study was to explore this hypothesis in greater detail in a sample of 215 undergraduate students. Individual differences in humor were measured using the Humor Styles Questionnaire (HSQ), and playfulness (i.e., low seriousness) was assessed using the trait version of the State-Trait Cheerfulness Inventory (STCI-T). Participants also completed a questionnaire about their substance use (smoking, alcohol, marijuana, cocaine). As predicted, affiliative humor, aggressive humor, and playfulness were significant predictors of greater substance use. Mediation analyses confirmed that the link between both affiliative and aggressive humor and substance use was mediated by seriousness, indicating that this association is due, at least in part, to high-humor individuals taking a less serious, more playful outlook on life. Overall, these results support the view that a sense of humor may be related to less healthy habits, at least in the domain of substance use.