Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S ribosomal RNA (rRNA) 3′ end maturation during late-stage 40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the genome-wide effects of eIF5B have not been studied at the single-nucleotide resolution in any organism, and 18S rRNA 3′ end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat stress acclimation by translational regulation, but its molecular function remained unknown. Here, we show that HOT3 is a late-stage ribosome biogenesis factor that facilitates 18S rRNA 3′ end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3′ end maturation or metabolism. We quantitatively defined processing hotspots and identified adenylation as the prevalent nontemplated RNA addition at the 3′ ends of pre-18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNA interference to generate RDR1- and DCL2/4-dependent risiRNAs mainly from a 3′ portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome-free fractions and were not responsible for the 18S rRNA maturation or translation initiation defects in hot3 . Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late 40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, messenger RNA (mRNA) translation initiation, and siRNA biogenesis in plants.