Over recent years, non-suicidal self-injury (NSSI) has been associated with emotion dysregulation. However, only a few quantitative studies have empirically investigated differences in emotion dysregulation among people who self-harm, while none have explored gender differences in this regard. Thus, this research study aimed to further examine the association between NSSI and emotion regulation deficits and strategies in young adults. A total of 201 participants (mean age = 21.82 years) were recruited from different support groups dedicated to NSSI and from health care centres and were divided into two groups: a control group (CG, n = 100, mean age = 21.92 years, comprised of 30% males) and an NSSI group (NSSIG, n = 101, mean age = 21.72 years, comprised of 16% males). All participants completed the Deliberate Self-Harm Inventory, the Difficulties in Emotion Regulation Scale, and the Emotion Regulation Questionnaire. The results showed that compared to the CG participants, those from the NSSIG had increased emotion regulation deficits, higher expressive suppression scores, and lower cognitive re-evaluation scores. Within the NSSIG, females had higher impulse control difficulties and limited access to emotion regulation strategies, while males had higher expressive suppression scores. Factors associated with NSSI also differed by gender. These results indicate the necessity to take gender into account when planning treatment, since treatment protocols must be adapted to the specific emotion regulation difficulties concerned.