This study aimed to determine the validity of the ActiGraph wGT3X-BT for step counts (SC) at five different body locations in laboratory settings. A total of 29 male adults participated in this study. The participants walked or ran for different speeds on the treadmill while wearing ActiGraph wGT3X-BT on their right wrist (RW), left wrist (LW), waist, right ankle (RA), and right upper arm (RUA). Both the video camera and each ActiGraph recorded SC simultaneously. The waist, RUA (6, 8, 10 km/hr and total), and RA (4 and 6 km/hr) showed small Mean Absolute Percentage Error (MAPE) values (<5%) and good agreement with Bland-Altman plots. However, the waist (2 and 4 km/hr), RUA (2 and 4 km/hr), RA (2, 8, 10 km/hr and total), RW, and LW showed high MAPE. In conclusion, the waist and RUA are valid for the total SC during walking and running in the laboratory setting.