The objective of this study was to develop a motion analysis system to evaluate relations between the activation of the lower limb muscles, the pedal force and the movement of the lower limb during the pedaling motion. To confirm the effectiveness of the proposed analysis system, we analyzed the pedaling motion of nine cyclists to identify their pedaling skill. In the experiments, cyclists were instructed to perform their pedaling technique at two pedaling rates (80-90 rpm and 110-120 rpm). Surface electromyography (EMG) signals was recorded from tensor of latae (TFL), rectus femoris (RF), hamstrings (HAM) and gastrocnemius (GAS), and pedal force was measured by a power meter (Pioneer Corporation). Furthermore, the lower limb motion during the pedaling was measured using a 3D motion capture system to investigate the relations among measured data as mentioned above. The experimental results show that the proposed analysis system reveals the pedaling skill of cyclists and the relations between the EMG data, the pedal force and the movement during the pedaling motion. In this paper, firstly, the activation section of the lower limb muscles during the pedaling was determined quantitatively. Secondly, Tukey-Kramer test was conducted to determine whether there are significant differences (p < 0.05) between the pedaling effectiveness of the three groups (elite group, amateur group and beginner group). Finally, the pedaling skill of the cyclists was revealed based on the analysis results of the measurement experiments.