The growing number of Mpox cases in China has posed a challenge to public health. The prevalence of men who have sex with men behaviors among students has been consistently increasing each year in China, accompanied by a high frequency of unprotected anal sex. As crowded places, schools are highly likely to cause an Mpox outbreak among students through long-term close contact. Understanding university students’ perceptions about Mpox and willingness to vaccinate play a vital role in implementing preventive measures in schools. This study aimed to assess knowledge, concerns, and vaccine acceptance toward Mpox among university students in North and Northeast China. A cross-sectional study was conducted among 3831 university students from seven universities in North and Northeast China between September 10 and September 25, 2023. This study found a relative insufficiency in Mpox knowledge among university students (71.60%), with less than half expressing concern about the Mpox outbreak (39.57%), and the majority exhibiting a positive attitude to vaccination (76.30%). Multivariate regression analysis revealed that a good knowledge level was associated with age, study discipline, education level, and a high level of concern about Mpox. Male, elderly, or highly educated participants had a low level of concern about Mpox. Participants with a high level of knowledge toward Mpox were more likely to have the vaccination willingness. This study might help governments and schools to understand students’ Mpox perceptions and vaccination intentions, enabling them to implement effective measures in addressing the issue of inadequate understanding regarding Mpox among university students.