Objective: To explore the safety of 5’-nucleotide in rats. Methods: Totally 96 rats were randomly divided into 6 groups and fed diet containing 5’-nucleotide at doses of 0, 0.01, 0.04, 0.16, 0.64 g/kg bw and 1.28 g/kg bw for 90 consecutive days. The changes of body weight, dietary intake amount, food utilization rate, blood pressure, blood routine, biochemical parameters, and pathological results of the rats were recorded at intervals of one week during the administration of 5’- nucleotide. Results: No obvious difference in total body weight gain, total dietary intake amount, food utilization rate and systolic blood pressure was observed between 5’-nucleotide treatment groups and control group. However, compared with the rats in control group, body weights of male rats in nucleotide treatment group exhibited a significant increase in the first 4 weeks (P < 0.05). Similarly, total food utilization rates of the rats treated with 5’-nucleotide at doses of 0.01, 0.04 g/kg bw and 0.16 g/kg bw exhibited significantly higher than that of control group in the first 2 weeks (P <0.05). In addition, compared with the control group, white blood cells, red blood cells, hemoglobin, platelets, total protein, albumin, alanine transaminasse, triglyceride of male rats also revealed a significant difference. However, all of these values were still in the normal range. No histopathological change was observed in kidney and other organs or tissues. Conclusion: It is safety to rats fed 5’-nucleotide at the dose of 0.01-1.28 g/kg bw.