Maturation of the 40S ribosomal subunit precursors in mammals mobilizes several non-ribosomal proteins, including the atypical protein kinase RioK2. Here, we have investigated the involvement of another member of the RIO kinase family, RioK3, in human ribosome biogenesis. RioK3 is a cytoplasmic protein that does not seem to shuttle between nucleus and cytoplasm via a Crm1-dependent mechanism as does RioK2 and which sediments with cytoplasmic 40S ribosomal particles in a sucrose gradient. When the small ribosomal subunit biogenesis is impaired by depletion of either rpS15, rpS19 or RioK2, a concomitant decrease in the amount of RioK3 is observed. Surprisingly, we observed a dramatic and specific increase in the levels of RioK3 when the biogenesis of the large ribosomal subunit is impaired. A fraction of RioK3 is associated with the non ribosomal pre-40S particle components hLtv1 and hEnp1 as well as with the 18S-E pre-rRNA indicating that it belongs to a bona fide cytoplasmic pre-40S particle. Finally, RioK3 depletion leads to an increase in the levels of the 21S rRNA precursor in the 18S rRNA production pathway. Altogether, our results strongly suggest that RioK3 is a novel cytoplasmic component of pre-40S pre-ribosomal particle(s) in human cells, required for normal processing of the 21S pre-rRNA.