Humans in the industrialized world spend a large amount of daily time in sedentary behavior. Since sedentariness negatively impacts a variety of psychophysiological outcomes, the identification of antecedents that lead to sedentary behavior is an important public health issue. In this context, mood, a central indicator for both psychological well‐being and mental health, is severely understudied. To investigate whether mood dimensions influence subsequent sedentary behavior, we assessed both constructs at baseline via questionnaires and via ambulatory assessment (AA) over 5 days in 92 university employees. We continuously measured sedentary behavior using accelerometers and assessed mood repeatedly 10 times each day on smartphone diaries. We employed multiple regression analyses to analyze between‐subject effects and multilevel modeling to analyze within‐subject effects. Higher momentary ratings of valence (P < .05) and energetic arousal (P < .01) predicted lower amounts of subsequent sedentary behavior, whereas higher ratings of calmness (P < .01) predicted higher amounts of subsequent sedentary behavior. The context moderated the effect of energetic arousal and calmness on sedentary behavior with increased effects in the home compared to the work context. Mood significantly predicted sedentary behavior on a within‐subject level but not on a between‐subject level. Preliminary evidence suggests that mood regulates sedentary behavior in everyday life. Time‐sensitive analyses, such as from moment to moment revealed an association between mood and sedentary behavior (within‐subject), whereas analyses between different individuals revealed no associations (between‐subject). These preliminary findings may inform multicomponent intervention strategies that target mood, to reduce sedentary behavior in daily life.