Background and aims Autotoxicity is a serious problem for continuous cropping of cucumber, it can limit its own growth and that of nearby cucumber plants and decrease yield and quality. Cinnamic acid (CA) derivatives are a class of autotoxins that can mimic autotoxic effects in cucumber. Methods We screened an Arabidopsis long hairpin RNA (lhRNA) library under CA-mimicked autotoxicity stress to obtain autotoxicity-resistant mutant lines. The glycerol-3-phosphate 2-O-acyltransferase 6 ( GPAT6 ) gene was found to be silenced in one resistant mutant line, and GPAT6 homologous gene in cucumber was cloned and named CuGPAT6. To test gene function quickly, a new tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) system was established in cucumber using a special agroinfiltration solution and whole cotyledonary nodes. Results Cucumber phytoene desaturase gene ( CuPDS ) and CuGPAT6 gene were successfully silenced with a newly established tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) system in cucumber showing photobleaching and decreased CuPDS expression in leaves; and silencing of CuGPAT6 enhanced the tolerance to the autotoxin stress mimicked by CA in cucumber. Conclusions It indicated that the alleviation of the damage caused by CA treatment was probably through maintaining the integrity of cellular structure, supporting cell vitality and promoting growth in roots, and increasing the integrate chloroplast and thylakoid numbers in leaves, which restored the growth and net photosynthesis rate of CA treated CuGPAT6 silenced plants to the similar level to those of control plants. The results also demonstrated that the newly established TRV mediated VIGS system is functional, rapid, convenient and highly efficient in cucumber.