Loss of TP53 function may contribute to 5-fluorouracil (5-FU) resistance in colorectal cancer since TP53-deficient cells may be unable to undergo apoptosis in response to 5-FU-induced DNA damage. 5-FU treatment of TP53-deficient cells would provide useful information on the apoptotic response to drug-induced DNA damage in the absence of TP53 and its transcriptional targets. We investigated apoptosis induction and cell cycle alterations in response to short-term treatment with two different 5-FU concentrations following siRNA-mediated knockdown of TP53 in the TP53-proficient HCT116 colon cancer cell line. We focused on high-dose 5-FU treatment to investigate the apoptotic phenotype in 5-FU-treated cultures since this dose resulted in apoptosis induction at 24 h of treatment, whereas clinically-relevant bolus 5-FU treatment of HCT116 cultures did not. Gene expression alterations were also assessed in 5-FU-treated HCT116 cultures using whole genome expression arrays. Compared to 5-FU-treated TP53-proficient HCT116 cultures, 5-FU-treated TP53-depleted HCT116 cultures showed lack of CDKN1A induction, decreased apoptotic levels, decreased FAS and TNFRSF10B transcript levels and cleaved PARP protein levels, G1/S transition arrests, decreased CCND1 protein levels, and smaller intra-S phase arrests. Alterations in gene expression in 5-FU-treated TP53-depleted HCT116 cultures confirmed previously-reported TP53 target genes and suggested potentially novel TP53 target genes (e.g. APOBEC3C, BIRC3, JMJD2B, LAMP3, MYO1E, PRRG1, SULF2, TACSTD2, TncRNA, ZFYVE20) that may play a role in mediating the 5-FU-induced DNA damage response in TP53-proficient cells. Abrogation of TP53 function in 5-FU-treated HCT116 cultures results in reduced apoptosis, TP53- and CDKN1A-independent G1/S phase arrests that may be protective against apoptosis, smaller intra-S phase arrests, and transcript level decreases of both reported TP53 target genes as well as potentially novel TP53 target genes.