The purpose of this study was to determine the muscle activities of the erector spinae (ES), gluteus maximus (Gmax), gluteus medius (Gmed), and the hamstring (HAM) and the ratios of Gmax/ES, Gmax/HAM, and Gmed/HAM during the prone heel squeeze (PHS) with different knee flexion angles (45˚, 90˚, and 135˚). Fifteen young and healthy subjects (8 men, 7 women) were recruited for the study. Surface electromyography signals were collected on ES, Gmax, Gmed, and HAM during PHS. A separate one-way analysis of variance with repeated measures was used to determine the significance of the muscle activities of ES, Gmax, Gmed, and HAM and the ratios of Gmax/ES, Gmax/HAM, and Gmed/HAM with different knee flexion angles during PHS. There was a significant increase in the Gmax activity at the knee flexion of 90˚ in comparison with that of the 45˚ (p=.016). There were significant increases in the Gmed activity at the knee flexion of 90˚ (p=.008) and 135˚ (p=.006) in comparison with that of the 45˚. There were significant decreases in the HAM activity at the knee flexion of 90˚ (p=.009) and 135˚ (p=.004) in comparison with that of the 45˚. There were significant increases in the Gmax/HAM muscle activity ratio at the knee flexion of 90˚ (p=.007) and 135˚ (p=.012) in comparison with that of the 45˚. There were significant increase in the Gmed/HAM muscle activity ratio at the knee flexion of 135˚ in comparison with that of the 45˚ (p=.008). The knee flexion of 90˚ during PHS can induce decreasing activity of HAM and increasing activity of Gmax, and the knee flexion of 135˚ during PHS can induce decreasing activity of HAM and increasing activity of Gmed. Hence, PHS with different knee flexion positions could be considered for the different target muscle.