Abstract Objectives Mastl is an essential kinase required for inhibition of the phosphatase activity directed toward Cdk1/cyclin B substrates during mitosis. Mastl phosphorylates two small evolutionarily conserved proteins, Arpp19 and Ensa converting them into strong inhibitors of PP2A-B55δ. Mastl-Arpp19/Ensa-PP2A regulatory pathway has been mainly studied in Xenopus egg extracts and Drosophila using biochemical and genetic approaches. Studies in mammalian cells and genetically modified mouse models have suggested distinct but important functions for Arpp19 and Ensa, in mitosis and S-phase, respectively. A detailed comparative analysis of the Arpp19 and Ensa functions in mammalian cells has not been performed. Methods We utilized Mastl conditional knockout (CKO) mouse embryonic fibroblasts (MEF), to investigate the roles of Mastl-Arpp19/Ensa-PP2A pathway components in mitosis and cellular proliferation. We used viral transduction for overexpression or silencing of these genes in conjunction with inducible genetic Mastl knockout to assess their roles in relation to each other. Results We show that, Arpp19 is expressed at significantly higher levels in MEFs in comparison to Ensa. Silencing of Arpp19, but not Ensa, results in reduced cellular proliferation. Overexpression of WT Arpp19 or its phosphomimetic mutant (S67D) partially restores mitosis arrest duration in Mastl knockout MEFs, however cellular proliferation block cannot be rescued. Silencing of B55δ expression has a similar outcome as Arpp19 overexpression, underscoring the opposite roles of these genes in mitosis. Conclusions Our results show that Arpp19 is the major Mastl substrate during mitotic division of MEFs. Ensa expression is low and it is not essential for cell cycle.