Vitamin A (retinol) is essential for normal fetal development, but the recommendation for maternal dietary intake (Retinol Activity Equivalent, RAE) does not differ for singleton vs. twin pregnancy, despite the limited evaluation of retinol status. Therefore, this study aimed to evaluate plasma retinol concentrations and deficiency status in mother–infant sets from singleton vs. twin pregnancies as well as maternal RAE intake. A total of 21 mother–infant sets were included (14 singleton, 7 twin). The HPLC and LC-MS/HS evaluated the plasma retinol concentration, and data were analyzed using the Mann–Whitney U test. Plasma retinol was significantly lower in twin vs. singleton pregnancies in both maternal (192.2 vs. 312.1 vs. mcg/L,p= 0.002) and umbilical cord (UC) samples (102.5 vs. 154.4 vs. mcg/L,p= 0.002). The prevalence of serum-defined vitamin A deficiency (VAD) <200.6 mcg/L was higher in twins vs. singletons for both maternal (57% vs. 7%,p= 0.031) and UC samples (100% vs. 0%,p< 0.001), despite a similar RAE intake (2178 vs. 1862 mcg/day,p= 0.603). Twin pregnancies demonstrated a higher likelihood of vitamin A deficiency in mothers, with an odds ratio of 17.3 (95% CI: 1.4 to 216.6). This study suggests twin pregnancy may be associated with VAD deficiency. Further research is needed to determine optimal maternal dietary recommendations during twin gestation.