More than 15% dietary protein has reportedly not led to significant muscle hypertrophy in normal growing rats. The aim of this study was to test whether a high protein (HP) diet affects myostatin (Mstn) synthesis in a rat gastrocnemius muscle. Twenty-four male Wistar rats (4-wk-old) were divided into three groups: 1) control diet (15% protein; 15P, n = 8), 2) the 25P group (25% protein, n = 8), and 3) the 35P group (35% protein, n = 8). After 3 wk of isoenergetic feedings, the Mstn level in skeletal muscles was determined using Northern and Western blotting analysis. After the experimental feeding, muscle masses were similar among groups. The 35P showed significant high expressions of Mstn both at mRNA and protein levels. Obtained results suggest that a high-protein diet leads to the high Mstn level to restrict muscle hypertrophy.