Background: Workplace interventions have utilized standing desks to reduce sedentary behavior. However, minimal information is available concerning factors that impact use of standing desks under real working conditions. Purpose: To qualitatively explore factors that influence standing desk use under real working conditions. Methods: Individual interviews were conducted with university employees currently using a standing desk using a semi-structured interview guide. Interviews were audio-recorded, transcribed verbatim, and coded for themes. Results: Participants (n = 37) identified factors impacting standing desk use across multiple socioecological levels. Intrapersonal-level barriers included forgetting to use the standing desk and pain or fatigue from standing; whereas, knowledge facilitated use. Social support facilitated use at the interpersonal level, but social norms were a barrier to standing. Access to standing desks and a wellness culture facilitated use at the institutional level, but the built environment was a barrier. Discussion: Findings from this study support a socioecological perspective of sedentary behavior and highlight the importance of incorporating intervention strategies at multiple levels to change sedentary behavior. Translation to Health Education Practice: The findings from this study may be used to develop multi-level strategies to initiate and maintain use of standing desks in order to overcome and prevent barriers impacting regular use.