Several studies in Europe and the United States have shown that sexual intercourse (SI) during pregnancy is not associated with preterm birth. However, it is unclear whether these findings apply to pregnant Japanese women. The aim of this prospective cohort study was to elucidate the influence of SI during pregnancy on preterm birth in Japan. A total of 182 women who underwent antenatal care and delivery were included in this study. The frequency of SI was assessed using a questionnaire, and its association with preterm birth was analyzed. The results showed that SI during pregnancy was associated with a significantly higher cumulative preterm birth rate (p= 0.018), which was more pronounced for SI more than once a week (p< 0.0001). Multivariate analysis showed that SI, bacterial vaginosis in the second trimester, previous preterm birth, and smoking during pregnancy were independent risk factors for preterm birth. The combination of SI and second trimester bacterial vaginosis was associated with a 60% preterm birth rate, whereas either factor alone was associated with a lower rate, suggesting a synergistic effect (p< 0.0001). Future studies are needed to investigate the effect of prohibiting SI in pregnant women with bacterial vaginosis on preterm birth.