Objective To assess the characteristics of twin fetal facial expressions at 30–33 + 6 weeks of gestation using four-dimensional (4D) ultrasound to clarify twin fetal brain development and maturation. Methods Frequencies of seven fetal facial expressions were studied using 4D ultrasound for 15 minutes in 30 singleton pregnancies and 18 twin pregnancies [four monochorionic diamniotic (MD) and 14 dichorionic diamniotic (DD) twins] at 30–33 + 6 weeks of gestation. Comparison of the frequency in each facial expression was performed between singleton and twin fetuses. Results Mouthing was the most frequent facial expression at 30–33 + 6 weeks of gestation, followed by blinking in twin and singleton fetuses. Both facial expressions were significantly more frequent than other expressions (P < 0.05). The frequencies of mouthing and scowling in twin fetuses were significantly lower than those in singleton fetuses, but there were no significant differences in the frequencies of the five other facial expressions between the fetal groups. Conclusion Our results suggest that restricted twin fetal behavior before 20 weeks of gestation may still affect the frequencies of twin facial expressions early in the third trimester of pregnancy. Moreover, the frequencies of facial expressions in twins are different from those of singleton fetuses.