Translation is a tightly regulated by numerous factors, among which mRNA length is a key factor to regulate translation efficiency. Compared with long mRNAs, short mRNAs are preferentially translated, which is called length-dependent translation. However, the underlying mechanism is largely unknown. Here we demonstrate that tRNA modification is a determinant of length-dependent translation. We found that loss of the evolutionarily conserved mcm5s2U modification of tRNA dramatically compromised immunity in Arabidopsis. Further analysis revealed that protein expression decreases with protein length in the absence of mcm5s2U modification, which is conserved in other eukaryotes. In consistence, tRNA modification affects the correlation between translation efficiency and mRNA length. Altogether, our study uncovers the link between tRNA modification and length-dependent translation, highlighting the importance of translation elongation in this process.