More research is needed to understand the different vulnerability profiles of university students who engage in non-suicidal self-injury (NSSI). This study sought to classify university students (n = 479; 83.8% female) aged 17–25 years (M = 18.77; SD = 1.42) who had engaged in NSSI within the past year into latent profiles based on their self-perceived difficulties in regulating both positive and negative emotions. Independent samples of students who had a past history of NSSI but had not self-injured within the previous year (n = 439; 82.9% females; Mage = 19.03, SD = 1.62) and who had no history of NSSI (n = 1551; 69.9% females; Mage = 19.02, SD = 1.55) were recruited for comparison purposes. Latent cluster analyses revealed three emotion regulation profiles within the NSSI sample—the Average Difficulties (47.4%), Dysregulated (33.0%), and Low Difficulties (19.6%) profiles—each of which differed meaningfully from both comparison samples on mean emotion regulation difficulties. Students across profiles also differed in their self-reported experiences with parents, particularly with fathers (pressure, antipathy, unresolved attachment, psychological control), and in the extent to which they felt alienated from parents. Lastly, students across profiles differed in the frequency, methods, functions, and addictive properties of their NSSI. Findings highlight that university students who self-injure experience distinct patterns of difficulties with emotion regulation, which are associated with variation in parent–child relational risk factors and NSSI outcomes.