Background: Ferula assa-foetida L has several therapeutic effects such as anticancer traits. The aim of this study was investigation of anticancer effect of Ferula assa-foetida gum alcoholic extract against CT-26, HT-29, SW742 and WiDr colorectal cancer cell lines. Methods: The hot extract was obtained by soxhlet using 70% ethanol solvent. Various cell lines were cultured in DMEM medium and in vitro anticancer effects using MTT assay was performed. A dilution (5, 10, 25, 50 and 100µg/mL) of the gum extract were subjected to cell lines in triplicate. and incubated for 24, 48 and 72hrs. The main phytochemicals within the gum extract were detected through Gas and mass chromatography. Results: cytotoxicity rates were observed at 50µg/mL and >100µg/mL, respectively for all cell lines. And morphological change was noticed after 72 hr. application of the gum extract on SW742 cell line. The compounds found in the Ferula assa-foetida L mostly included oxygen-containing sesquiterpenes (45.25%) and sesquiterpenes (30.45%). Conclusion: the viability decreasing was observed for all examined colorectal adenocarcinoma cell lines including SW742, CT-26, HT-29 and WiDr lines depend on time and concentration of the applied extract of F. assa-foetida gum. The potent cytotoxicity was observed in 50-100 µg/Ml. Furthermore, a morphological change was detected after 72 hr. on SW742. A high percentages of anticancer compounds which were detected within the gum extract supports the idea of purifying these components and considering them in cancer drugs, phytochemistry.