Introduction: Yogurt is mixed with spinach in some countries. Spinach can be a probable dietary source of Nitrate and Nitrite, and further N-nitrosamine. Methods: The present study aimed to investigate the effect of inoculation of Lactobacillus acidophilus on the level of nitrite and nitrate in “yogurt containing spinach” (YS) during refrigeration in for 21 days. 4 types of yogurts including yogurt (Y), probiotic yogurt (PY), YS, and probiotic YS (PYS) were produced and kept at 4 °C for 21 days. Their nitrite and nitrate levels were measured by HPLC on days 1, 7, 14, and 21. Results: There was no significant difference between the mean concentrations of nitrate and nitrite of Y and PY and between YS and PYS. Although the effect of refrigeration at 4 °C for 21 days was not significant on the nitrate amount of Y and PY, it was close to a significant level in YS and PYS. It also caused a significant increase in the nitrite content of all yogurts. This study showed that Lactobacillus acidophilus bacteria cannot affect the amount of nitrate and nitrite in yogurt or YS at 4°C. Conclusion: Increasing the nitrite content of samples may enhance the possibility of nitrosamine formation. It is suggested that those who are at risk for all malformations caused by nitrites should not keep YS for a long time.  Keywords: nitrate; nitrite; Lactobacillus acidophilus; probiotic yogurt containing spinach; HPLC