Children conceived through intracytoplasmic sperm injection (ICSI) have been reported to have a higher risk of many abnormalities and disorders, including autism and intellectual disability, which may be due to bypassing of the natural sperm selection process during ICSI. Zona pellucida (ZP)-bound spermatozoa (ZPBS) have normal morphology and nuclear DNA. Using these spermatozoa for ICSI results in better outcomes compared with conventional ICSI. However, differences besides morphology that exist between sperm selected by ZP and by an embryologist and whether these differences affect the risk of autism in offspring after ICSI are unclear. To explore these questions, we compared genome-wide DNA methylation profiles between ZPBS and manually selected spermatozoa (MSS)using single-cell bisulfite sequencing. Global DNA methylation levels were significantly lower in ZPBS than in MSS. Using gene ontology (GO) analysis, genes overlapping differentially methylated regions (DMRs) were enriched in biological processes involving neurogenesis. Furthermore, we found that 47.8% of autism candidate genes were associated with DMRs, compared with 37.1% of matched background genes (P<0.001). This was mainly because of the high proportion of autism candidate genes with bivalent chromatin structure. In conclusion, bivalent chromatin structure results in large differences in the methylation of autism genes between MSS and ZPBS. ICSI using MSS, which increases the risk of methylation mutations compared with ZPBS, may lead to a higher risk of autism in offspring.