Mealworms (Tenebrio molitor larvae) are considered a highly nutritious and sustainable protein source. Bioactive peptides produced from protein hydrolysis have recently gained interest as alternative chemotherapeutic agents due to their potential to improve physiological activities. Muscle atrophy, wasting or loss of muscle mass, is a prevalent problem limiting physical activity and reducing the overall quality of life. Nonetheless, there are currently few investigations on natural agents preventing muscle atrophy. In this study, two potential peptides, Phe-Asp-Lys-Tyr (FDKY) and Phe-Asp-Arg-Leu (FDRL), were purified and identified from mealworm protein hydrolysate (MPH), and their inhibitory potentials of myostatin, a major negative regulator of skeletal muscle, in C2C12 cells were determined. Mealworm protein isolate (MPI) was obtained at alkaline pH, and MPH was prepared with flavourzyme and alcalase. MPH was separated by ultrafiltration membranes (3kDa and 10kDa). The MT (10kDa<), which has the most significant myostatin inhibitory potential, was fractionated (D1-D4) by anion exchange chromatography. The D1 was re-chromatographed by RP-HPLC and separated into five fractions (C1–5). Subsequently, the C3 was subjected to LC-MS/MS, and seven peptides were identified. Luciferase activity was evaluated to investigate the myostatin promoter activity, and RT-PCR was performed to determine the mRNA levels of myostatin. Consequently, D1 and C3, which have the most significant potential for myostatin inhibition in each fractionation step, had the highest branched-chain to total amino acid ratios (16.98% and 15.12%, respectively). Among all the peptides, only the treatment of FDKY (14.41%) and FDRL (15.74%) significantly lowered the luciferase activity compared to the control group (no treatment) in a dose-dependent manner. FDKY treatment also decreased myostatin mRNA expression levels (31%) compared to the control group. These results suggest that the bioactive peptides from MPH could be an attractive natural agent that can be used as a drug or functional food ingredient to prevent muscle atrophy.