Updated publication reference for PubMed record(s): 33292386. Background: The presence of nuclear mitochondrial DNA (numtDNA) has been reported within several nuclear genomes. Next to mitochondrial protein coding genes, numtDNA sequences also encode for mitochondrial tRNA genes. However, the biological roles of numtDNA, remain elusive. Results: Employing in silico analysis we identify 281 mitochondrial tRNA homologs in the human genome, which we term nimtRNAs (nuclear intronic mitochondrial-derived tRNAs), being contained within introns of 76 nuclear host genes. Despite base changes in nimtRNAs when compared to their mtRNA homologs, a canonical tRNA cloverleaf structure is maintained. To address potential functions of intronic nimtRNAs, we insert them into introns of constitutive and alternative splicing reporters and demonstrate that nimtRNAs promote pre-mRNA splicing, dependent on number and positioning of nimtRNA genes and splice site recognition efficiency. A mutational analysis reveals that the nimtRNA cloverleaf structure is required for the observed splicing increase. Utilizing a CRISPR/Cas9 approach we show that a partial deletion of a single endogenous nimtRNALys within intron 28 of the PPFIBP1 gene decreases inclusion of the downstream located exon 29 of the PPFIBP1 mRNA. By employing a pull-down approach followed by mass spectrometry, a 3’-splice site associated protein network is identified, including KHDRBS1, which we show directly interacts with nimtRNATyr by an electrophoretic mobility shift assay. Conclusions: We propose that nimtRNAs, along with associated protein factors, can act as a novel class of intronic splicing regulatory elements in the human genome by participating in the regulation of splicing.