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Despite the time-honored tradition in teacher stress research of treating class size as a job-related stressor, the underlying premise that class size directly impacts teachers’ stress reactions remains unsubstantiated. In this experimental study targeted at pre-service teachers, we utilized a standardized virtual reality (VR) classroom to examine whether class size directly affects both the physiological (heart rate) and psychological (subjective rating) stress reactions among 65 pre-service teachers. Results from linear mixed-effects modeling (LMM) shown that average heart rate (HR) and subjective stress ratings were both significantly higher in the large class size condition. These findings may contribute to a better understanding of the effects of classroom features on pre-service teachers’ emotional experiences and long-term well-being.