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 have been no reports yet regarding the genetic dissection of the geminivirus-plant interaction. Here, a high-throughput approach was developed to screen Arabidopsis activation-tagged mutants which are resistant to geminivirus Beet severe curly top virus (BSCTV) 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 the 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 affects BSCTV infection by activating the SA pathway. This study thus provides a new approach to study of the geminivirus-host interaction.