Background: Non-suicidal self-injury (NSSI) behavior among college students is a focus of attention in current society. In the information era, the Internet serves as a public health concern and as an effective pathway for prevention. In order to reduce NSSI behavior, we explore its influence factors, especially the relations between neuroticism, emotion regulation (ER), depression, and NSSI behavior. Methods: A total of 450 college students were surveyed with the Big Five Inventory-2, Emotion Regulation Questionnaire, Self-Rating Depression Scale, and Adolescent Non-Suicidal Self-Injury Assessment Questionnaire. Results: Regression analysis showed that neuroticism significantly negatively predicted emotion regulation, while it positively predicted depression and NSSI. Multiple mediation modeling demonstrated that neuroticism and emotion regulation had no significant direct effects on NSSI. However, neuroticism could indirectly affect NSSI through four pathways of multiple mediating effects, including depression, cognitive reappraisal-depression, expressive suppression-depression, and cognitive reappraisal-expressive suppression-depression. Conclusions: Neuroticism positively predicts depression and NSSI behavior, and affects NSSI through the mediating effect of ER and depression. Therefore, amelioration of neuroticism from the perspectives of emotion regulation and depression is recommended, so as to reduce NSSI behavior among college students with highly neurotic personalities.