The southern root-knot nematode, Meloidogyne incognita, is one of the most prevalent and damaging plant-parasitic nematodes in the world and causes serious damages to agricultural production. We cloned a mitochondrial ATP synthase b subunit gene fragment of M. incognita (MiASB) based on the nematode genomics prediction. By soaking in the MiASB dsRNA solution, the hatching of RNAi treated eggs was reduced by 60% compared to negative control and by 64% compared to untreated control. Mortality of RNAi treated second stage juvenile (J2) was 8.6 times higher than that of negative control and 26 times higher than the untreated control. Inoculating the RNAi treated egg masses and J2 to tomato seedlings showed the pathogencity was significantly reduced. For the RNAi treated egg masses, the amount of root galls on silence treated seedlings was reduced by 92% compared to that on the negative control seedlings, and reduced by 93% compared to that on untreated control seedlings. For the treated J2, the amount of root galls on silence treated seedlings was reduced by 83% and 86% compared to negative and untreated control seedlings, respectively. The study revealed the MiASB silence had a positive effect on prevention and control of root-knot nematode disease, and also showed that the MiASB may be involved in the pathogenesis of nematode, which provided new ideas and ways to the research of nematode pathology and nematode disease control.