microRNAs (miRNAs) are short RNA molecules, which post-transcriptionally regulate other genes. They are generated from genome-encoded single-stranded hairpin structures, by a set of RNA-processing enzymes. miRNAs are highly important for gene regulation and involved in a variety of physiological and developmental processes. Recently, both the miRNA processing enzymes and expressed miRNAs were discovered in unicellular relatives of animals, in a group called Ichthyosporea. This was the first evidence for miRNAs in close relatives of animals, and challenged the view that animal miRNA was an innovation early in the evolution of animals. So far, the evidence from miRNAs in ichthyosporeans is only from a single genus, Sphaeroforma, and only very few miRNAs were detected based on limited sequence data. Nothing is known about their functional roles. Therefore, I used small RNA data from additional ichthyosporean species, Abeoforma whisleri and Pirum gemmata, in addition to Sphaeroforma species to identify novel miRNAs. I also investigate the functional roles of these miRNAs by performing target prediction. Furthermore, I have performed degradome sequencing in Sphaeroforma arctica to investigate whether there are genes that are subjected to miRNA-induced cleavage. I identify 180, 74, 105, 13, 16, and 19 miRNAs in A. whisleri, P. gemmata, S. arctica, S. sirkka, S. tapetis, and S. gastrica respectively by mapping small RNA reads against draft genomes. I have for the first time identified miRNAs in two ichthyosporean species A. whisleri and P. gemmata outside Sphaeroforma, as well as several novel miRNAs among Sphaeroforma sp. I find that the majority of these miRNAs are not conserved between the ichthyosporean lineages, although a few miRNAs are conserved within Sphaeroforma. For almost all the miRNAs, there are potentially a large number of sites in the transcriptome with nearly full complementarity where the miRNAs have the possibility to bind and regulate genes. In addition, by sequencing cleaved mRNAs in S. arctica I was able to prove that 18 gene transcripts which had in fact been cleaved by miRNAs at the predicted cleavage sites. This strongly supports a role of miRNAs in S. arctica involved in target cleavage, a process which exists in both plants and basal animals. This supports the hypothesis that the ancestral function of the animal miRNAs is cleavage of target mRNAs.