Considering the limitations of intraportal transplantation (Tx), we sought to establish an alternative approach for it-transplanting islets onto the liver surface (LS) by optimizing adipose tissue-derived stem cell (ADSC) co-Tx procedures with a gelatin hydrogel nonwoven fabric (GHNF). In the in vivo study, we examined the use of the GHNF, the effectiveness of islet covering materials, and preferred procedures for ADSC co-Tx using a syngeneic rat model. Immunohistochemical staining was performed to evaluate the extracellular matrix (ECM) expression and angiogenesis. In the in vitro study, we analyzed the culture supernatants to identify crucial factors secreted from ADSCs in different ADSC co-Tx procedures. It was shown that the GHNF should be used to cover the islets but not to embed internally (encapsulate) them. Utilization of the GHNF in LS Tx resulted in significantly better glucose changes (P = 0.0002) and cure rate of diabetic recipients (P = 0.0003) than the use of a common adhesion barrier. Although neovascularization was comparable among groups, ECM reconstitution tended to be higher when the GHNF was used. ADSC co-Tx further enhanced ECM reconstitution only when ADSCs were cultured in the GHNF before islet Tx. Leptin, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and several chemokines were identified as candidate factors for enhancing ECM reconstitution (P < 0.001). The inhibition assay using antagonist suggested that leptin might be at least in part responsible for the difference in transplant efficiency in distinct ADSC co-Tx methods. This study showed that the GHNF effectively improved the outcomes of LS islet Tx, mainly due to ECM reconstitution around the islets. Furthermore, we established a novel method of LS islet Tx by combining a GHNF with ADSCs, which is equally effective as intraportal Tx.