Background: Daptomycin stands as a key IV antibiotic in treating MRSA infections. However, patients facing challenges with difficult venous access require alternative administration routes. This study aimed to evaluate the pharmacokinetic (PK) profile and safety of subcutaneous (SC) daptomycin. Patients and methods: In a two-period, two-treatment, single-blind crossover Phase I trial (ClinicalTrials.gov NCT04434300), participants with no medical history received daptomycin (10 mg/kg) both IV and SC in a random order, with a minimum 2 week washout period together with matched placebo (NaCl 0.9%). Blood samples collected over 24 h facilitated PK comparison. Monte Carlo simulations assessed the PTA for various dosing regimens. Adverse events were graded according to Common Terminology Criteria for Adverse Events(CTCAE) v5.0. Results: Twelve participants (aged 30.9 ± 24.4 years; 9 male,75%) were included. SC daptomycin exhibited delayed (median Tmax 0.5 h for IV versus 4 h for SC) and lower peak concentration than IV (Cmax = 132.2 ± 16.0 μg/mL for IV versus 57.3 ± 8.6 μg/mL for SC; P < 0.001). SC AUC0-24 (937.3 ± 102.5 μg·h/mL) was significantly lower (P = 0.005) than IV AUC0-24 (1056.3 ± 123.5 μg·h/mL) but was deemed bioequivalent. PTA demonstrated target AUC0-24 attainment for 100% of simulated individuals, for both 8 and 10 mg/kg/24 h SC regimens. Adverse events (AEs) related to SC daptomycin were more frequent than for SC placebo (25 versus 13, P = 0.016). No serious AEs were reported. Conclusions: Single-dose SC daptomycin infusion proved to be safe, exhibiting a bioequivalent AUC0-24 compared with the IV route. The SC route emerges as a potential and effective alternative when IV administration is not possible.