Abstract: This study explored the effects of silver nanoparticles (AgNPs) on root growth, cellular integrity, and negative gravitropism in Arabidopsis thaliana (L.) Heynh.. Results revealed that: (1) Treatment with 30 mg/L AgNPs led to a significant accumulation of Ag in roots, surpassing the levels observed in roots treated with 0.12 mg/L Ag⁺ (equivalent to the Ag⁺ released from 30 mg/L AgNPs). (2) Exposure to 30 mg/L AgNPs markedly inhibited the number and length of root hairs. Transcriptome sequencing and RT-qPCR analyses indicated that 30 mg/L AgNPs suppressed the expression of key genes associated with root hair cell development, including AtRHS12, AtRHS14, AtCOW1, and AtMRH12. (3) Both 30 mg/L AgNPs and 30 mg/L Ag⁺ induced root callus formation and caused significant root cell damage in A. thaliana. However, no significant root damage was observed in plants treated with 0.12 mg/L Ag⁺, suggesting that the Ag⁺ released from AgNPs was insufficient to cause cellular damage. (4) Exposure to 30 mg/L AgNPs induced a spiral growth pattern in the main root, contrasting with the leftward growth induced by 30 mg/L and 0.12 mg/L Ag⁺. Transcriptome sequencing and RT-qPCR analyses revealed that both 30 mg/L AgNPs and 30 mg/L Ag⁺ significantly down-regulated ethylene-regulated genes such as AtERS1, AtETR2, AtERF1, AtERF11, and AtEBP, while up-regulating the key ethylene synthesis gene AtACS7. These findings suggest that AgNPs and Ag⁺ may influence negative gravitropism in A. thaliana roots by modulating ethylene signaling pathways and ethylene biosynthesis. In conclusion, 30 mg/L AgNPs inhibit root hair growth, induce root cell damage, and influence gravitropic bending in A. thaliana roots through the ethylene signaling pathway. The observed toxic effects are likely attributable to the intrinsic properties of the nanoparticles.