This cross-sectional study investigated the associations of business type with the prevalence of metabolic syndrome (MetS) and lifestyle characteristics among workers in small-and medium-sized enterprises. In total, data from 167,736 workers (114,746 men and 52,990 women) who participated in health checkups in 2013 were analyzed using multilevel logistic regression models. The odds ratios (ORs) of having MetS, defined based on the criteria of the joint interim statement, were significantly higher in employees of transportation businesses (reference OR =1) than in other business types among men (OR: 0.67-0.85) and similar result was observed among women (OR: 0.70-0.88). The prevalence of a smoking habit was significantly higher in transportation workers than in employees of other businesses for both men and women. Furthermore, male transportation workers were more likely to skip breakfast, engage in <1 h/day of walking, walk at a slower speed, and eat dinner just before going to bed. Female transportation workers were more likely to have gained 10 kg since the age of 20 years. In conclusion, the prevalence of MetS was higher in transportation workers than in workers from other businesses; the associated risk factors may also vary by sex. To effectively promote public health, the labor environment, such as the business type, should be considered.