Objective: To determine the impact of progressive anemia of prematurity on cerebral regional saturation (C-rSO2) in preterm infants and identify the hemoglobin threshold below which a critical decrease (>2SD below the mean) in C-rSO2 occurs. Study design: In a cohort of infants born ≤30 weeks EGA, weekly C-rSO2 data were prospectively collected from the second week of life through 36 weeks post-menstrual age (PMA). Clinically obtained hemoglobin values were noted at the time of recording. Recordings were excluded if they were of insufficient duration (<1 h) or if the hemoglobin was not measured within 7 days. Statistical analysis was performed using a linear mixed effects-model and ROC analysis. ROC analysis was used to determine the threshold of anemia, where C-rSO2 critically decreased >2SD below the mean normative value (<55%) in preterm infants. Results: In total 253 recordings from 68 infants (mean EGA 26.9 ± 2.1 weeks, BW 1025 ± 287 g, 49% male) were included. Approximately 29 out of 68 infants (43%) were transfused during hospitalization. Mixed-model statistical analysis adjusting for EGA, BW, and PMA revealed a significant association between decreasing hemoglobin and C-rSO2 (p < 0.01) in transfusion-naive infants but not in transfused infants. In the transfusion naive group, using ROC analysis demonstrated a threshold hemoglobin of 9.5 g/dL (AUC 0.81, p < 0.01) for critical cerebral desaturation in preterm infants. Conclusions: In transfusion-naive preterm infants, worsening anemia was associated with a progressive decrease in cerebral saturations. Analysis identified a threshold hemoglobin of 9.5 g/dL below which C-rSO2 dropped >2SD below the mean.