Pathogen reduction technology (PRT) may improve the safety of RBCs for transfusion. As the Czech Republic considers PRT, we asked what effects riboflavin and UV light PRT pre-freezing has on the post-thaw recovery and properties of cryopreserved RBCs (CRBCs) after deglycerolization and liquid storage. 24 Group O whole blood (WB) units were leukoreduced and then treated with riboflavin and UV light PRT (Mirasol, Terumo BCT, USA) before cryopreservation (T-CRBC); 20 similarly-collected units were untreated controls (C-CRBC). Units were processed to RBCs and then cryopreserved with 40% glycerol (wt/vol), frozen at -80°C, stored >118 days, reconstituted as deglycerolized RBC units in AS-3, and stored at 4 ± 2°C for 21 days. One treated unit sustained massive hemolysis during the post-thaw wash process and was removed from data analysis. The remaining units were assessed pre-PRT, post-PRT, and post-thaw-wash on days 0, 7, 14, and 21 for hematocrit, volume, hemoglobin per transfusion unit, pH, % hemolysis, hemoglobin in the supernatant, potassium, phosphorus, NH3 , osmolality, ATP, and 2,3-diphosphoglycerate. PRT with leukoreduction caused a 5% loss of RBC followed by a 24% freeze-thaw-wash related loss for a total 28% loss but treated units contained an average of 45 g of hemoglobin, meeting European Union guidelines for CRBC. T-CRBCs displayed higher post-wash hemolysis, potassium, and ammonia concentrations, and lower ATP at the end of storage. Cryopreserved RBCs from Riboflavin and UV light-treated WB meet the criteria for clinical use for 7 days after thawing and provide additional protection against infectious threats.