This study aims to investigate the mediation role of cyber-ostracism to explain the relationship between perceived stress and emotional well-being among college students. We adopted a computer-based experiment, "Ostracism Online," randomly assigned participants to cyber-ostracized (n = 66) and cyber-included (n = 79) groups. The program simulated social media platforms, and participants created self-accounts and gave out Likes. Participants in cyber-ostracized group were manipulated to receive one Like, whereas those in cyber-included group received nine Likes. Before the experiment, perceived stress was measured, and cyber-ostracism and emotional well-being were measured after the ostracism manipulation. The findings revealed that cyber-ostracism fully mediates the relationship between perceived stress and emotional well-being. The results can be used to develop an online intervention for cyber-inclusion enhancement to reduce the negative emotions of stressed college students.