Looking for useful and motivational strategies for promoting healthy habits and improving cognitive functioning in young populations, the aim of the present study was to determine if a single bout of high-intensity interval exercise could stimulate mood and working memory in college students. A total of 25 male subjects (mean ± SD, age: 21.7 ± 2.1 years; height: 1.77 ± 0.06 m; weight: 72.6 ± 8.4 kg; body mass index: 23.1 ± 1.4 kg/m; VO: 47.1 ± 9.3 mL/kg/min) participated voluntarily in this study. Participants underwent a high-intensity interval exercise consisting of 10 × 1 min of cycling at VO power output. The Profile of Mood States (POMS) questionnaire and Digit Span Test (DST) were administered at three assessment time points: (a) pre-intervention assessment, (b) post-intervention assessment, and (c) 30 min post-intervention. The mood states decreased significantly after exercise; however, a significant increase in mood was found after 30 min of recovery. A significant post-exercise increase in DST performance was observed; moreover, DST scores obtained 30 min after exercise remained higher than those assessed pre-exercise. In conclusion, a single bout of HIIT induces acute positive changes in mood states in male college students and seems to be a powerful stimulus for cognitive functioning.