Tobacco smoking is endocrine-disrupting and may interfere with vitamin D endocrine systems (VDES), but supporting evidence is limited and inconsistent. Also, there is a lack of evidence on whether the association between tobacco smoke exposure and VD levels exhibit temporal variation. Data from the National Health and Nutrition Examination Survey was used to evaluate the association between tobacco smoke exposure and VD levels among US general participants from 2001 to 2014. We examined the linear association between serum cotinine and 25(OH)D concentrations, as well as relationship between tobacco smoke exposure categories (active, passive, non-smoking) with VD status (deficiency, inadequacy, sufficiency, intoxication), and assessed whether specific gender, age (3-11, 12-19, 20-59, ≥ 60 years), ethnicity/race, or body mass index (BMI) groups were disproportionately impacted. During 2001-2004, a decrease in both serum cotinine and passive smoking prevalence was observed, with a stabilized active smoking rate. The estimates for the association between tobacco smoke exposure and suboptimal VD levels increased over the study period. Overall results indicated that serum cotinine was negatively associated with 25(OH)D in all participants. Tobacco smoke exposure, including both active and passive smoking exposure, was associated with increased risk of VD deficiency. Moreover, active smoking was additionally related to enhanced risk of VD inadequacy. These associations showed some age and gender differences, with consistent and stronger associations observed in female adults. In contrast, effects of tobacco smoke exposure on VD levels were mostly negative or non-significant among children and adolescents aged 3-19 years. The percentage of US general population with active smoking exposure stabilized over the 14-year period and was still high. Tobacco smoke exposure may disrupt vitamin D levels with an increasing temporal trend in the risk. Our results also provided initial evidence of smoking exposure on VD intoxication, which needs to be further verified. Convincing studies have linked tobacco use exposure, to dysfunctional VDES accompanied with declined serum levels of VD metabolites. However, evidence on the association between tobacco smoke exposure and VD status was rather limited and inconsistent, and there were no researches to date that estimated the temporal variation of the association as well as the effects of smoke exposure on VD intoxication. This study analyzed national survey data, to evaluate the temporal trends in effects of tobacco smoke exposure on VD levels over a decade, and to comprehensively assess the impacts of tobacco smoke exposure on VD levels across specific subgroups. The evidence suggests that the prevalence of active smoking exposure stabilized over the 14-year period and was still high. Moreover, tobacco smoke exposure may disrupt vitamin D levels among general population, with an increasing temporal trend and age-, gender-differences in risk.