In this study, the effects of pulse electric field (PEF) treatment on the tenderization of beef semitendinosus muscle were investigated. An adjustable PEF chamber was designed to make direct contact with the surface of the beef sample without water as the PEF-transmitting medium. PEF treatment was conducted with electric field strengths between 0.5 and 2.0 kV/cm. The pulse width and pulse number were fixed as 30 μs and 100 pulses, respectively. The impedance spectrum of PEF-treated beef indicated that PEF treatments induced structural changes in beef muscle, and the degree of the structural changes was dependent on the strength of the electric field. Cutting force, hardness, and chewiness were significantly decreased at 2.0 kV/cm (35, 37, and 34%, respectively) (p < 0.05). Troponin-T was more degraded by PEF treatment at 2.0 kV/cm intensity (being degraded by 90%). The fresh quality factors such as color and lipid oxidation were retained under a certain level of PEF intensity (1.0 kV/cm). These findings suggest that PEF treatment could tenderize beef texture while retaining its fresh quality.