It is important to establish experimental animal techniques that are applicable to the newborn and infant phases for nutrition and pharmacological studies. Breeding technology using the artificial suckling method without breast milk is very effective for the study of newborn nutrition. Using this method, we separated newborn mice from dams within 48 h of birth and provided them with artificial milk. We evaluated mouse anxiety levels after early postnatal maternal separation. Artificially reared mice were subjected to elevated plus-maze tests to assess emotional behavior at 9 weeks of age. Artificially reared mice showed a significantly lower frequency of entries and dipping into the open arms of the maze compared with dam-reared mice. This result indicates that the anxiety level of artificially reared mice was higher than that of dam-reared mice. Moreover, the concentration of monoamines in the brain was determined after the behavioral experiment. The hippocampal norepinephrine, serotonin, and 5-hydroxyindoleacetic acid levels in the artificially reared mice were significantly higher than those of the dam-reared mice. These results suggest that maternal-offspring interactions are extremely important for the emotional development of newborn infants during the lactation period. In future studies, it is necessary to consider the environmental factors and conditions that minimize the influence of artificial rearing on emotional behavior.