Objectives: Emotional stimuli differently affect working memory (WM) performance. As sleep deprivation has a known impact on both emotion and WM our aim was to investigate how one night without sleep affects emotional WM performance. Methods: Healthy subjects (n = 56; age 18–30 years) were randomized to a total sleep deprivation (TSD) or a rested control (RC) condition. Subjects rated their affective state and performed a 1 and a 3-back WM task consisting of neutral, positive and negative pictures at 3 pm or 6 pm (balanced) the day after sleep manipulation. Accuracy (d’) and target response time (RT) were used as outcomes. Results: In the TSD condition, subjects rated themselves as less positive (P = 0.006) but not more negative than in the RC condition. In the WM task, TSD had a detrimental effect on accuracy (P = 0.03) regardless of difficulty. Moreover, accuracy was higher in the 1-back than in the 3-back (P < 0.001) and higher for neutral compared to both negative and positive stimuli (Ps < 0.05). RT was faster for positive compared to negative and neutral stimuli (Ps < 0.05). The latter effect was particularly pronounced in the TSD condition as shown by a condition*valence interaction (P < 0.03). Conclusions: One night of total sleep loss impaired emotional WM accuracy. Noticeable, RT was faster for positive stimuli compared to negative and neutral stimuli. This effect was particularly pronounced after sleep loss. This suggests that sleep loss strengthens the opposing effects of positive and negative stimuli on WM performance, possibly due to increased emotion reactivity.