Extraction of upper bicuspids have been anecdotally blamed to increase the vertical gingival display (VGD) anteriorly. However, the extraction may be needed in some cases in order to correct the underlying orthodontic problem. Objectives: To investigate and compare vertical gingival display (VGD) changes associated with upper (first vs second) premolars extraction during orthodontic treatment. Design: A prospective clinical trial. Setting: Postgraduate dental teaching clinics at Jordan University of Science and Technology (JUST). Sample population: Sixty orthodontic patients were included in the study. They were treated with upper first or second premolars extraction according to the underlying problem and the individualized treatment plan of each patient. Records (radiographs, study casts and clinical photographs) were taken for all subjects pre- and post- orthodontic treatment. Outcome measures: Pre- and post-treatment VGD, lip length in static and dynamic positions and the amount of upper teeth retractions were recorded. The paired and the independent t- test were used to detect differences within/between groups. Factors affecting VGD were investigated using backward stepwise linear regression analysis. In both static and dynamic captures, VGD increased after orthodontic treatment in both premolars extraction groups. Pre- and post-treatment variables differed significantly in groups 1 and 2. VGD changes were similar in both treatment groups. A significant association was found between VGD change during orthodontic treatment and upper canine retraction (P<0.001), pre-treatment ANB angle (P<0.01) and upper incisor retraction(P<0.05). The amount of anterior VGD increases after upper premolars extraction. The increase in VGD after first and second premolars extractions was comparable. The increase in VGD after orthodontic treatment is associated with the amount of canine retraction, pre-treatment ANB and the amount of incisor retraction.