ABSTRACT: Heat shock protein 70 (HSP70) usually functions in maintaining protein homeostasis, and is also involved in regulating plant immunity, but mediation of plant-parasitic nematode resistance by HSP70s has rarely been reported. In this work, two high similarity soybean GmHSP70s [GmHSP70-13a (Glyma.13g254900) and GmHSP70-15a (Glyma.15g059900)] were studied in soybean cyst nematode (SCN) resistance, which is a devastating pathogen in soybean. Both GmHSP70-13a and GmHSP70-15a were significantly suppressed in resistant soybeans, compared to susceptible soybeans, at 0 day and 3 days post inoculation (dpi) of SCN. In soybean hairy roots, CRISPR/Cas9-editing of these two genes resulted in enhanced SCN resistance; accordingly, overexpressing either GmHSP70-13a or GmHSP70-15a suppressed SCN resistance. Both GmHSP70-13a and GmHSP70-15a interacted with GmSHMT08, which is one major SCN-resistant protein in soybeans and a key enzyme in one-carbon metabolism pathway. Compared to 0 dpi of SCN, at 3 dpi, GmHSP70-13a overexpression did not alter and GmHSP70-15a overexpression significantly suppressed, in contrast, the control remarkably promoted, GmSHMT08 expression in soybean. Furthermore, either GmHSP70-13a or GmHSP70-15a overexpression abnormally down-regulated expression of 4 one-carbon metabolism-associated genes (Glyma.06g235500, Glyma.12g015300, Glyma.15g071000, and Glyma.15g071200). Taken together, both GmHSP70-13a and GmHSP70-15a negatively mediated soybean SCN resistance likely by interacting with GmSHMT08, leading to GmSHMT08 suppression, along with impact of one-carbon metabolism pathway in soybean. This work provides 2 GmHSP70s gene-editing targets for soybean SCN resistance breeding.