Few efforts have been made regarding the optimization of porcine small intestinal submucosa (SIS) to improve its biocompatibility. This study aims to evaluate the effect of SIS degassing on the promotion of cell attachment and wound healing. The degassed SIS was evaluated in vitro and in vivo, compared with the nondegassed SIS control. In the cell sheet reattachment model, the reattached cell sheet coverage was significantly higher in the degassed SIS group than in the nondegassed group. Cell sheet viability was also significantly higher in the SIS group than in the control group. In vivo studies showed that the tracheal defect repaired by the degassed SIS patch showed enhanced healing and reductions in fibrosis and luminal stenosis compared to the nondegassed SIS control group, with the thickness of the transplanted grafts in the degassed SIS group significantly lower than those in the control group (346.82 ± 28.02 µm vs. 771.29 ± 20.41 µm, p < 0.05). Degassing the SIS mesh significantly promoted cell sheet attachment and wound healing by reducing luminal fibrosis and stenosis compared to the nondegassed control SIS. The results suggest that the degassing processing might be a simple and effective way to improve the biocompatibility of SIS.