Objectives: Increasing bilateral gluteus medius co-activation has been identified as one of the most important factors in developing low back pain due to prolonged standing in healthy people. This study aims to investigate the impact of an anti-fatigue mat on the bilateral gluteus medius co-activation pattern and to report the low back pain subjectively in 2 different standing positions on the normal rigid surface and on the anti-fatigue mat. Material and Methods: While carrying out an easy simulated profession, 16 participants who had no low back pain background were requested to stand for 2 h in each position, with and without using the anti-fatigue floor mat, respectively. At the beginning of standing process and at every 15 min until the time of 120 min lapses, electric activities for the bilateral gluteus medius co-activation and subjective pain level in low back area were collected by the surface electromyogeraphy (EMG) and the visual analogue scale (VAS), respectively in each position. Results: The obtained findings revealed that the anti-fatigue mat significantly decreased subjective pain level in low back area among 15 participants (p < 0.05). However, there was objectively no significant difference in the bilateral gluteus medius co-activation pattern among the participants between the position 1 and the position 2 (p > 0.05). The findings obtained under this study related to the impact of the anti-fatigue mat upon the low back pain based on the increase of > 10 mm on the VAS threshold, which showed that this intervention had no significant impact upon decreasing the number of patients suffering from the low back pain and also minimizing the bilateral gluteus medius co-activation in both pain developer groups (p > 0.05). However, 73% of the participants preferred to apply it. Conclusions: It seems that the anti-fatigue mat may be useful in reducing the low back pain although it objectively didn’t significantly change the gluteus medius co-activation pattern related to the low back pain.