Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys⁵⁷ and Lys⁴⁰¹ of human PDI in vitro. Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys⁵⁷ and Lys⁴⁰¹ was assessed by in vitro treatment using acetylsalicylic acid (aspirin). A total of 28 acetyllysine residues were identified, including acLys⁵⁷ and acLys⁴⁰¹. The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.