N-methylformamide (NMF) is an anti-proliferative, differentiating agent studied in several cell lines as well as in preclinical and clinical trials, whose mechanisms of action are still unclear. 9-Hydroxystearic acid (9-HSA) is an endogenous product of lipid peroxidation recently identified as a new histone deacetylase 1 inhibitor. Both agents show the same anti-proliferative effects by arresting colon cancer cell growth in G0/G1. We addressed two questions. (i) Do they act by regulating G0/G1 checkpoint proteins? (ii) Does 9-HSA have differentiating effects comparable to those of NMF? The effects of NMF and 9-HSA on growth, differentiation and invasiveness of HT29, a colon cancer cell line, have been compared by using immunoprecipitation analysis, confocal microscopy, enzyme assays and invasiveness tests. The results show that the G1 arrest caused by NMF is a cell cycle exit due to p27 induction, whereas 9-HSA has no effect on the induction of this inhibitor. Evidence is presented that the arrest in early G0/G1 induced by 9-HSA is associated with the conversion of HT29 characteristics to those of a more benign phenotype, whereas the arrest in the late G1 in response to NMF is not followed by a decrease in tumorigenicity. The failure of NMF in cancer therapy indicates that both anti-proliferative and differentiating characteristics are required for an anti-tumoral agent to be effective.