Pegunigalsidase alfa is a newly approved drug for the treatment of Fabry disease, designed to increase the plasma half-life and reduce immunogenicity of infused α-galactosidase A (AGAL). We provide the first comprehensive pharmacokinetic and immunogenic data apart from industry-initiated studies.Pharmacokinetics of pegunigalsidase alfa, amino acid, and polyethylene glycol (PEG)-specific antibodies and immune complexes were measured in treated patients (11 switched, two naïve). Measurements were performed in serum samples drawn directly before and after infusions over three to ten consecutive infusions. Only three patients started directly with 1.0 mg/kg body weight.No infusion-associated reactions were reported under pegunigalsidase alfa during the observation. Patients without pre-existing neutralizing anti-AGAL antibodies showed high enzymatic AGAL peak activities and sustained AGAL serum concentrations until the next infusion, which was not observed in those with neutralizing anti-AGAL antibodies. Nine (69.2%) patients presented with pre-existing anti-PEG antibodies (IgG or IgM), which seemed to have no impact on pharmacokinetics during the observation. No new anti-PEG or anti-AGAL antibody formation was observed after treatment initiation. Three (75.0%) patients with pre-existing neutralizing anti-AGAL antibodies showed a titer increase and one (25.0%) patient a decrease. In patients with anti-AGAL antibodies (n = 4) immune-complex formation was detected.The pharmacokinetics of pegunigalsidase alfa show different profiles depending on the presence of pre-existing neutralizing antibodies, with reduced plasma half-life and peak enzyme activity after infusion in patients with antibodies. The clinical significance of a reduced pegunigalsidase alfa half-life and the formation of immune complexes in antibody-positive patients needs to be analyzed in future studies.