Twin pregnancy differs considerably from singleton pregnancy in many aspects and it is unknown how serum NT-proBNP level behaves in women with twin pregnancies. Serum NT-proBNP levels were determined longitudinally at gestational weeks (GW) 24 and 35 in normotensive women with 13 twin and 99 singleton pregnancies. The effects of maternal demographic characteristics on NT-proBNP levels were also analyzed. The serum NT-proBNP levels (pg/mL) in twin pregnancies, which were not different from those in singleton pregnancies at 24 GW (26 ± 15 vs. 40±27, respectively, P = 0.0718), increased significantly (P = 0.0038) and were significantly higher than those in singleton pregnancies at 35 GW (72 ± 49 vs. 34 ± 24, P < 0.0001). In the analysis including women with singleton pregnancies, the serum levels of NT-proBNP at 35 GW were significantly inversely correlated with pre-pregnancy body mass index (BMI, kg/m 2 ) and were significantly higher in nulliparous than multiparous women. Thus, women with twin pregnancy were likely to exhibit an increase in serum NT-proBNP levels in the late stage of pregnancy, especially in lean and nulliparous women. The relative greater blood volume expansion occurring in twin than in singleton pregnancies was considered to be responsible for this phenomenon.