Non-coding (nc) RNAs are important regulators of developmental genes, and essential for the modification of cellular DNA and chromatin through a process known as RNA interference (RNAi). The mediators of RNAi can be in the form of short double stranded (ds) RNAs, micro (mi) RNAs or small interfering (si) RNAs. miRNAs are involved in a translation repression pathway that inhibits protein translation in mRNA targets. Comparative genomic screens have revealed conserved regulatory non-coding sequences, which assist to predict the function of endogenous miRNAs. Only a few comparative studies include bovine genomic sequence, and RNAi has yet to be applied in bovine genome functional screens. siRNAs target homologous mRNAs for degradation, and thereby, silence specific genes. The use of synthetic siRNAs facilitates the elucidation of gene pathways by specific gene knockdown. A survey of the literature identifies a small number of reports using RNAi to examine immune pathways in bovine cell lines; however, they do not target genes involved in specific production traits. Applications of RNAi to elucidate bovine immune pathways for relevant bacterial and parasite diseases are yet to be reported. The inhibition of viral replication using RNAi has been demonstrated with bovine RNA viruses such as pestivirus and foot and mouth disease virus signifying the potential of RNAi as an antiviral therapeutic. RNAi approaches combined with genome data for protozoan parasites, insects and nematodes, will expedite the identification of novel targets for the treatment and prevention of economically important parasitic infections. This review will examine the approaches used in mammalian RNAi research, the current status of its applications to livestock systems and will discuss potential applications in beef cattle programs.