Treatment of painful neuromas remains a challenge and the mechanism of neuroma-associated pain is not yet fully understood. In this study, we aimed to observe the expression of alpha smooth muscle actin (α-SMA) in traumatic neuromas and to investigate its possible roles in the cause of neuropathic pain in a rat model. The rat sciatic nerve was used and the experiment was divided into two parts. In part I, our results showed significantly higher levels of α-SMA and the pain marker c-fos in the autotomy group than in the no-autotomy group. In part II, the expression of α-SMA in neuromas was down- and up-regulated using SB-431542 and GW9662, respectively. A significant correlation between autotomy scores and the expression level of α-SMA was found (R = 0.957; p < 0.001) and the expression level of α-SMA was positively related to the autotomy scores (R(2) = 0.915, p < 0.001). We concluded that the expression of α-SMA plays certain roles in the neuroma-associated pain, either as a direct cause of pain or as an indirect marker of existence of local mechanical stimuli. Our findings may provide new insights into the development of new treatment modalities for the management of intractable painful neuromas.