A novel colloidal creatine formulation has been suggested to be superior to creatine monohydrate for improving plasma creatine levels after single-dose administration in animal research. However, if colloidal creatine supremely affects creatine levels in the skeletal muscle and blood of healthy humans remains currently unknown. This double blind, cross-over, randomized, creatine monohydrate-controlled pilot trial evaluated serum and skeletal muscle levels of creatine before and during single-dose colloidal creatine (Creaphil®) administration in five healthy men. Five healthy volunteers aged 24.0 ± 1.7 years (weight 79.2 ± 7.4 kg; height 181.0 ± 5.7 cm) were supplemented with single dose of 5 g of colloidal creatine or creatine monohydrate, with skeletal muscle and serum levels of creatine monitored at baseline and during post-administration for 10 h in five intervals. At each assessment at the lab, the participants completed magnetic resonance spectroscopy for right vastus medialis muscle and clinical chemistry studies. Colloidal formulation resulted in a more powerful rise in vastus medialis creatine levels comparing to creatine monohydrate, as evaluated with the area under the concentration-time curve calculation (108.5 ± 15.9 mM · h vs. 103.3 ± 15.1 mM · h; P = 0.01). This was accompanied by a significantly higher serum creatine level after colloidal creatine administration at 30 min post-administration (158.4 ± 21.2 μmol/L vs. 121.0 ± 38.9 μmol/L; P = 0.05). No participant reported any adverse events during the trial. These results suggest that colloidal creatine is a preferred and safe alternative to creatine monohydrate for improved creatine bioavailability after single-dose administration. However, more studies are needed to evaluate its effectiveness and safety after long-term supplementation.