Promoter hypermethylation preventing expression of the RAS association domain family 1 isoform A (RASSF1A) gene product is among the most abundant epigenetic deregulations in human cancer. Restoration of RASSF1A inhibits tumor cell growth in vitro and in murine xenograft models. Rassf1a-deficient mice feature increased spontaneous and carcinogen-induced tumor formation. Mechanistically, RASSF1A impacts on several cellular functions, such as microtubule dynamics, migration, proliferation and apoptosis; however, its tumor-suppressive mechanism is incompletely understood. To study the functional consequences of RASSF1A expression in human cancer cells, we made use of a doxycycline-inducible expression system and a RASSF1A-deficient lung cancer cell line. We observed that RASSF1A induces cell cycle arrest in G1 phase and senescence in vitro and in tumors established in immune-deficient mice. RASSF1A-mediated growth inhibition was accompanied by upregulation of the Cyclin-dependent kinase (CDK) inhibitor p21Cip1/Waf1 and proceeded independently of p53, p14Arf and p16Ink4a. Loss of p21Cip1/Waf1 or co-expression of the human papilloma virus (HPV) 16 oncoprotein E7 were found to override RASSF1A-induced cell cycle arrest and senescence. Conditional RASSF1A impacted on mitogen-activated protein kinase (MAPK) and protein kinase B/Akt (Akt) signaling to upregulate p21Cip1/Waf1 and to facilitate its nuclear localization. In summary, RASSF1A can mediate cell cycle arrest and senescence in human cancer cells by p53-independent regulation of p21Cip1/Waf1.