Whether orthodontic treatment can change the preferred chewing side (PCS) is unknown. This study examined (1) if the PCS changes after orthodontic treatment and (2) which factors contribute to this change. Two hundred fifty patients who visited the orthodontic clinic at Tokyo Medical and Dental University Hospital between 2017 and 2020 were included in the study. Mandibular kinesiograph (MKG) was taken at pre- and post-treatment, and PCS was determined. Patients who showed a change in PCS to the opposite side and those who showed no change in PCS at post-treatment were pooled into the PCS-changed and PCS-unchanged groups, respectively. The demographic, clinical, and cephalometric parameters were compared between the groups. Significant factors associated with changes in were of age < 20 years at the beginning of orthodontic treatment (odds ratio (OR), 2.00), maximum lateral mandibular movement to PCS ≥ 10.0 mm at pre-treatment (OR, 6.51), and change in occlusal canting of ≥1.0° (OR, 2.72). The predicted probability of change in PCS was 13.2%, 36.0%, and 67.5% for no factor, one factor, and two factors associated with PCS change, respectively. Orthodontic treatment may change PCS due to patient age, maximum lateral mandibular movement to PCS, and change in occlusal canting.