Small ribozymes cleave their RNA phosphodiester backbone by catalyzing a transphosphorylation reaction wherein a specific O2' functions as the nucleophile. While deprotonation of this alcohol through its acidification would increase its nucleophilicity, little is known about the pKa of this O2' in small ribozymes, in part because high pKa's are not readily accessible experimentally. Herein, we turn to molecular dynamics to calculate the pKa of the nucleophilic O2' in the hairpin ribozyme and to study interactions within the active site that may impact its value. We estimate the pKa of the nucleophilic O2' in the wild-type hairpin ribozyme to be 18.5 ± 0.8, which is higher than the reference compound, and identify a correlation between proper positioning of the O2' for nucleophilic attack and elevation of its pKa. We find that monovalent ions may play a role in depression of the O2' pKa, while the exocyclic amine appears to be important for organizing the ribozyme active site. Overall, this study suggests that the pKa of the O2' is raised in the ground state and lowers during the course of the reaction owing to positioning and metal ion interactions.