The purpose of this study was to determine the immediate effect of three types of shoe insoles, on electromyography activity of selected lower extremity muscles during running on treadmill. The electromyography activity of the rectus femoris, vastus lateralis, vastus medialis, biceps femoris, tibialis anterior and gastrocnemius lateralis muscles of 14 male athletes were recorded in non-insole, wearing soft, semi-rigid and rigid insoles conditions during running on treadmill. Electromyography activity of the rectus femoris muscle in the loading phase was lower in soft insole condition than other conditions. In the pre-swing/early swing phase, there was difference of the rectus femoris muscle activity in the soft/non-insole and soft/semi-rigid conditions. In this phase, the biceps femoris muscle had different activity in the semi-stiff/non-insole and semi-rigid/rigid conditions. In the mid-swing phase, there was a difference in the activity of the rectus femoris and the tibialis anterior muscles in the non-insole/semi-rigid condition, and the gastrocnemius lateralis muscle in the non-insole condition with other conditions. There was difference also for biceps femoris muscle activity in the semi-rigid/non-insole condition in the pre-activation phase. The vastus lateralis and gastrocnemius lateralis muscles activity in the soft insole condition was lower than that of the semi-rigid condition. It seems that the intensity and contraction pattern of the muscles change while using different shoe insoles with different variety of stiffness during running. It could be useful in choosing a shoe insole with a suitable stiffness material level for clinical and training purposes.