miRNAs are small non-coding RNA molecules that are capable of regulating hundreds of genes and are involved in many, if not, all biological pathways. We sought to investigate the utility of these molecules as potential engineering targets in CHO cells and subsequently to identify the mechanisms by which they mediate any observed effect. Previous miRNA profiling studies conducted in our laboratory identified several candidate miRNAs associated with cell proliferation. Functional assessment of several of these miRNAs using mimic and inhibitor molecules showed that miR-30e-5p, miR-29a, miR-10a and let-7e impacted on growth and, despite contrasting results between assays and cell lines could be good candidates to manipulate three main bioprocess-relevant CHO properties, i.e. cell proliferation, cell survival and protein production/secretion. The functional importance of miR-24 in CHO cell proliferation was also demonstrated. Transient overexpression or knockdown of the three members of the miR-23a~27a~24-2 cluster proved to be a better approach than manipulation of the individual miRNAs due to their presumably cooperative roles in regulating cell growth. Furthermore, stable cell lines expressing antisense transcripts to simultaneously knockdown the miR-23~27a~24-2 cluster members showed increased proliferation of up to 71% compared to control lines. The anti-proliferative role of miR-7 was verified using mimics in several different CHO cell lines in serum-free medium and we found that the arrest of cell growth upon pm-7 transfection was associated with accumulation in G1 of the cell cycle without induction of apoptosis. Expression profiling and experimental validation revealed that miR-7 targets key regulators of the G1 to S phase transition, including Skp2, Myc and p27Kip1 to temporarily arrest cell growth in the G1 phase. The down-regulation via miR-7 of critical pro-apoptotic regulators, including p53, as well as DNA repair factors such as Rad54L, with the concomitant up-regulation of anti-apoptotic factors like p-Akt, promoted cell survival while arrested in G1. Finally, we successfully extended the longevity of culture and consequently improved CHO cell proliferation by 3.27-fold as well as viability by 28.9% respectively in batch-fed culture using an antisense ‘miRNA sponge’ approach to stably knockdown endogenous miR-7.