Summary Geminiviruses include a large number of single-stranded DNA viruses that are emerging as useful tools to dissect many fundamental processes in plant hosts. However, there has been no report yet regarding genetic dissection of the geminivirus-plant interaction. Here, a high-throughput approach was developed to screen Arabidopsis activation tagged mutants which are resistant against geminivirus BSCTV (Beet severe curly top virus) infection. A mutant, lsb1 (less susceptible to BSCTV 1), was identified, in which BSCTV replication was impaired and BSCTV infectivity was reduced. We found that the three genes closest to the T-DNA were up-regulated in lsb1, and the phenotypes of lsb1 could only be recapitulated by the overexpression of GDU3 (GLUTAMINE DUMPER 3), a gene implicated in amino acid transport. We further demonstrated that activation of LSB1/GDU3 increased expression of components in the salicylic acid (SA) pathway, which is known to counter geminivirus infection, including the upstream regulator ACD6. These data indicate that up-regulation of LSB1/GDU3 impacts BSCTV infection by activating the SA pathway. Together, this study provides a new approach to study the geminivirus-host interaction.