In type 2 diabetes mellitus (T2DM), decreased autonomic activation and heightened negative emotions may worsen glycemic control. This study investigated the effects of heart rate variability biofeedback (HRVB) on autonomic activation, negative emotions, diabetes self-care, and glycemic control in patients with T2DM. A total of 61 participants with T2DM were assigned to either the HRVB group (n = 30; 62.67 ± 7.28 years; 14 females) or the control group (n = 31; 63.39 ± 6.96 years; 14 females). Both groups received the treatment as usual, and the HRVB group received 60 min of HRVB sessions weekly for 6 weeks. Participants completed psychological questionnaires, a resting electrocardiogram (ECG), and breathing rate assessments at pre- and post-tests. Heart rate variability (HRV) indices were derived from ECG data, and glycated hemoglobin (HbA1c) levels were collected from the electronic medical records. The analysis revealed significant Group × Time interaction effects on HRV indices, breathing rate, depression symptoms, and diabetes self-care behavior. The HRVB group demonstrated higher HRV indices, lower breathing rate, and improved diabetes self-care behavior compared to the control group. Moreover, the HRVB group showed enhanced HRV indices and diabetes self-care behavior, as well as reduced breathing rate and depression in the post-test compared to the pre-test. However, there was no significant interaction effect on HbA1c levels. Six sessions of HRVB proved effective as a complementary therapy for T2DM, enhancing HRV indices, alleviating depressive symptoms, and promoting better diabetes self-care behaviors.