The direct link between inflammatory bowel diseases and colorectal cancer is well documented. Previous studies have reported that some lactic acid bacterial strains could inhibit colon cancer progression however; the exact molecules involved have not yet been identified. So, in the current study, we illustrated the tumor suppressive effects of the newly identified Lactobacillus acidophilus DSMZ 20079 cell-free pentasaccharide against colon cancer cells. The chemical structure of the purified pentasaccharide was investigated by MALDI-TOF mass spectrum, 1D and 2D Nuclear Magnetic Resonance (NMR). The anticancer potentiality of the purified pentasaccharide against both Human colon cancer (CaCo-2) and Human breast cancer (MCF7) cell lines with its safety usage pattern were evaluated using cytotoxicity, annexin V quantification and BrdU incorporation assays. Also, the immunomodulatory effects of the identified compound were quantified on both LPS-induced PBMC cell model and cancer cells with monitoring the immunophenotyping of T and dendritic cell surface marker. At molecular level, the alteration in gene expression of both inflammatory and apoptotic pathways were quantified upon pentasaccharide-cellular treatment by RTqPCR. The obtained data of the spectroscopic analysis, confirmed the structure of the newly extracted pentasaccharide; (LA-EPS-20079) to be: α-D-Glc (1→2)α-L-Fuc(1→4) α-D-GlcA(1→2) α-D-GlcA(1→2) α-D-GlcA. This pentasaccharide, recorded safe dose on normal mammalian cells ranged from 2 to 5 mg/ml with cancer cells selectivity index, ranged of 1.96-51.3. Upon CaCo-2 cell treatment with the non-toxic dose of LA-EPS-20079, the inhibition percentage in CaCo-2 cellular viability, reached 80.65 with an increase in the ratio of the apoptotic cells in sub-G0/G1 cell cycle phase. Also, this pentasaccharide showed potentialities to up-regulate the expression of IKbα, P53 and TGF genes. The anticancer potentialities of LA-EPS-20079 oligosaccharides against human colon cancer represented through its regulatory effects on both apoptotic and NF-κB inflammatory pathways.