The aim of this study was to characterize the electromyographic (EMG) profile of tibialis posterior during barefoot walking in order to establish a reference database for neutral foot posture. Fifteen participants had their foot posture screened using the six-item Foot Posture Index. Bipolar intramuscular electrodes were inserted into tibialis posterior and peroneus longus utilizing ultrasound guidance. Surface electrodes were placed over medial gastrocnemius, peroneus brevis and tibialis anterior. EMG and footswitch gait characteristics were recorded whilst participants completed 10 barefoot walking trials. Individual and grand ensemble averages were used to characterize the intensity profiles for each muscle. Results indicated that for most of the participants, tibialis posterior displayed two bursts of EMG activity, with the first burst during the initial contact phase and the second burst during midstance. However, there was significant variability between participants. The grand ensemble average for tibialis posterior was comparable to peroneus longus which displayed similar temporal and intensity characteristics. It is suggested that this may reflect a synergistic relationship between these muscles during stance phase, although this was not consistent for all participants. Further research is required to determine if this relationship is altered in abnormal foot posture and whether it is clinically important. In conclusion, the EMG profile of tibialis posterior during the gait cycle appeared to be highly variable among participants. However, the authors believe that EMG findings from the participants with neutral foot posture in this study may be used for comparison to EMG patterns in people with abnormal foot posture and individuals affected by musculoskeletal disease.