It has been reported that office environment is an important determinant of physical activity (PA) and sedentary behavior (SB) in office workers. However, the effect of changes in office environment (office renovation) is unclear. The purpose of this study was to examine PA, SB, and space utilization changes among office workers in response to office renovation. This study was a natural experiment at three offices of a single company in Tokyo, Japan. The participants were, 13 workers from one office in the renovation group (mean age: 37.9 ± 10.8 years, percentage of females: 23.1%) and 29 from two offices in the control group (mean age: 42.3 ± 11.2 years, percentage of females: 31.0%). In the renovation, introduction of activity-based working (ABW) and installation of height-adjustable desks (HAD) were adopted. The ABW office was designed to provide various shared workstations, enabling the workers to choose workstations depending on their task or mood. Accelerometer measurement and object detection method using artificial intelligence (AI) technology for video images were used to assess behavior and space utilization before and after the renovation. Two weeks after the renovation, significant improvements in SB (pre-to post-renovation improvements: 346.8 ± 28.6 to 321.2 ± 17.8 min/working-hours) and PA (total PA: 173.2 ± 28.6 to 198.8 ± 17.8 min/working-hours; and light-intensity PA: 130.4 ± 27.1 to 150.7 ± 31.0 min/working-hours) were observed. In addition, the results of the object detection analysis showed that the central aisle of the office and shared HAD workstations near the entrance or window were utilized more frequently than the other spaces. This study suggested that office renovation could improve SB and PA immediately after the renovation. Moreover, utilized spaces and HAD workstations could play an important role to enhance employees' activity in an ABW office.