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Objective: Gastric cancer is a common cause of death worldwide. It is highly prevalent in Iran, especially in the north. It appears that vitamin D (Vit D) has a role in preventing cancer cells from proliferating. Thus, this study aimed to determine the role of Vit D in the effectiveness of chemotherapeutic drugs on cellular categories. Patients and Methods: The MTT assay, flow cytometry, and TNF-α cytokine concentration measurement were employed in this experimental study to evaluate the cytotoxicity effects of Vit D on cellular categories of gastric cancer such as MKN 45 (NC: C615), KATO III (NC: C640), and AGS (NC: C131). The statistical t-test was utilized in SPSS 16 to analyze data and determine the mean of 24 h cell proliferation and cytokine generation. Results: This study investigated the toxicity effect using the MTT assay based on combined treatment with Vit D and chemotherapeutic drugs. The results indicated a statistically significant difference (p<0.05) between cancer cells and normal cells in the base dose (μM50). Regarding the tested cellular categories, AGS and KATO III showed the highest (83%, 57%) and lowest (2%, 38%) cytotoxicity means of combined interventions (Vit D with chemotherapeutic drugs), respectively. Moreover, despite the decreased cell viability in different concentrations of Vit D, this decrease was not statistically significant (p>0.05). According to the flow cytometry results, the apoptosis percentages of 5FU group (60.7%) and 5FU-Vit D group (76.05%) were higher than that of the control group (25.66%). Conclusions: According to the research results, Vit D decreased the viability of gastric cancer cells and infused the apoptosis process into them. It also prevented the proliferation of cancer cells, something which can be used in the future to treat patients after conducting animal studies and clinical trials.