Introduction <p>Hyaluronidase has been used as an adjuvant to facilitate subcutaneous drug delivery by degrading hyaluronic acid, a viscoelastic barrier in subcutaneous tissue. However, traditional animal-derived hyaluronidases raise safety concerns, including risk of anaphylaxis and zoonoses. This study aimed to evaluate the safety, tolerability, and pharmacokinetics of ALT-BB4, a novel recombinant hyaluronidase derived from human hyaluronidase PH20, in healthy adults.</p> Methods <p>This first-in-human, multicenter, randomized, double-blinded, placebo-controlled phase 1 study included 244 participants who received single intradermal or subcutaneous injections of ALT-BB4 or placebo. The study was conducted in three parts: part I assessed drug allergy reactions, part II-A evaluated pharmacokinetics, and part II-B assessed safety and tolerability.</p> Results <p>Intradermal injection of ALT-BB4 exhibited a low incidence of drug allergy reactions (0.4%), with no significant difference compared with placebo (<i>p</i> = 0.317). Subcutaneous injection of ALT-BB4 resulted in more frequent injection site treatment emergent adverse events (TEAEs) compared with placebo (16.9% versus 0%; <i>p</i> &lt; 0.001). All injection site TEAEs were mild, self-resolving, and did not require treatment. Systemic TEAEs were less frequent in the ALT-BB4 group compared with placebo (0.7% versus 5.6%; <i>p</i> = 0.045), and no serious adverse events were reported. Notably, no antidrug antibodies were detected. Pharmacokinetic analysis revealed minimal systemic absorption of ALT-BB4.</p> Conclusions <p>Both intradermal and subcutaneous injection of ALT-BB4 demonstrated excellent safety and tolerability, supporting its potential as a promising alternative to traditional animal-derived hyaluronidases. Future studies are warranted to confirm these findings in broader clinical settings.</p> Trial Registration <p>ClinicalTrials.gov identifier, NCT05232175.</p>