This study investigated multi-generational reproductive and developmental toxicity of dietary nucleotides (NTs) during lactation in rats with the purpose of augmenting currently available safety data. Weanling rats (F0) were fed NTs at doses of 0.01%, 0.04%, 0.16%, 0.64%, and 1.28% (wt/wt for 90 days and then mated in a 1:1 pattern. The F1 generation rats were fed NTs for 90 days after weaning. The process was repeated until F3 rats were born. We measured the physical and neural development landmarks during lactation in the F3 generation and observed the signs of maternal rats during the gestation, parturition, and lactation periods. No significant differences in either physical or neural developmental landmarks were observed between the control and NT groups. No clinically adverse effects were found in the maternal rats at any point in gestation, parturition, or lactation. There were no alterations in the indicators of parturition. No significant differences were observed between controls and NT-treated rats fed four kinds of nucleotides in F0 milk on PND 21. In conclusion, NTs in doses of up to 1.28% did not cause any adverse effects in either the maternal or filial individuals.were shown non-toxicity of NTs during lactation. The NOAEL is considered to be 1.28%.