The DNA methylation of human offspring can change due to the use of assisted reproductive technology (ART). In order to find the differentially methylated regions (DMRs) in ART newborns, cord blood maternal cell contamination and parent DNA methylation background, which will add noise to the real difference, must be removed. We analyzed newborns’ heel blood from six families to identify the DMRs between ART and natural pregnancy newborns, and the genetic model of methylation was explored, meanwhile we analyzed 32 samples of umbilical cord blood of infants born with ART and those of normal pregnancy to confirm which differences are consistent with cord blood data. The DNA methylation level was lower in ART-assisted offspring at the whole genome-wide level. Differentially methylated sites, DMRs, and cord blood differentially expressed genes were enriched in the important pathways of the immune system and nervous system, the genetic patterns of DNA methylation could be changed in the ART group. A total of three imprinted genes and 28 housekeeping genes which were involved in the nervous and immune systems were significant different between the two groups, six of them were detected both in heel blood and cord blood. We concluded that there is an ART-specific DNA methylation pattern involved in neuro- and immune-system pathways of human ART neonates, providing an epigenetic basis for the potential long-term health risks in ART-conceived neonates.