The RAD9 protein assembles with RAD1 and HUS1 in the 9-1-1 DNA damage sensor complex (RAD9-HUS1-RAD1) which plays a crucial role in the activation of the DNA damage checkpoints by recruiting repair enzymes to DNA lesions and by initiating a cell cycle arrest. Why human cells express two RAD9 proteins, RAD9A and RAD9B, which share significant similarity is still very enigmatic. Moreover, both genes encode several splice variants with unknown function. While RAD9A is intensively studied, very little information is available about RAD9B. To get a deeper insight into the biology of RAD9B, two stable HEK293 cell lines were constructed which either over-express full-length human RAD9B-002 (417aa) or its N-terminally truncated splice variant RAD9B-001 (345aa) from an inducible promotor. These studies were complemented by the expression analysis of all five RAD9B splice variants at mRNA level in human tissues. This study revealed for the first time specific activities of the two RAD9B variants. While both proteins target the CHK2-p21 signalling module, which regulates cell cycle progression from G1 into S phase and the response to DNA damage in G1/S, they do affect CHK2 kinase and the cell cycle regulator p21WAF1/CIP1 in distinct ways. Elevated protein levels of full-length RAD9B initiate cell death in G1, cause the moderate hyperposphorylation of CHK2 and delay the degradation of p21 in the presence of oxidative stress. On the contrary, high levels of the N-terminally truncated variant RAD9B-001 only transiently block G1-S transition, cause a strong hyper-phosphosphorylation of CHK2 and delay p21 degradation in the response to UV irradiation. While the shorter variant may block p21 degradation in a PCNA-dependent manner in S phase, the full-length protein may act through the anaphase promoting complex (APC) when cells exit mitosis. The work provides also evidence that the hyper-phosphorylation of CHK2 is not dependent on the normal upstream kinase ATM, but on the mitotic regulator MPS1/TTK kinase. Dissecting this novel regulatory network formed by the two splice variants of the human RAD9B gene may help to understand why RAD9B expression levels change in several human cancers including testicular cancer, myelogenous leukaemia, ovarian epithelial carcinoma and cervical carcinoma (HeLa) cells.