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DNA methylation plays important roles through the methyl-CpG-binding domain (MBD) to realize epigenetic modifications. Thirteen AtMBD proteins have been identified from the <i>Arabidopsis thaliana</i> genome, but the functions of some members are unclear. AtMBD3 was found to be highly expressed in pollen and seeds and it preferably binds methylated CG, CHG, and unmethylated DNA sequences. Then, two mutant alleles at the AtMBD3 locus were obtained in order to further explore its function using CRISPR/Cas9. When compared with 92.17% mature pollen production in the wild type, significantly lower percentages of 84.31% and 78.91% were observed in the <i>mbd3-1</i> and <i>mbd3-2</i> mutants, respectively. About 16–21% of pollen from the <i>mbd3</i> mutants suffered a collapse in reproductive transmission, whereas the other pollen was found to be normal. After pollination, about 16% and 24% of <i>mbd3-1</i> and <i>mbd3-2</i> mutant seeds underwent early or late abortion, respectively. Among all the late abortion seeds in <i>mbd3-2</i> plants, 25% of the abnormal seeds were at the globular stage, 31.25% were at the transition stage, and 43.75% were at the heart stage. A transcriptome analysis of the seeds found 950 upregulated genes and 1128 downregulated genes between wild type and <i>mbd3-2</i> mutants. Some transcriptional factors involved in embryo development were selected to be expressed, and we found significant differences between wild type and <i>mbd3</i> mutants, such as <i>WOXs</i>, <i>CUC1, AIB4,</i> and <i>RGL3</i>. Furthermore, we found a gene that is specifically expressed in pollen, named <i>PBL6</i>. PBL6 was found to directly interact with AtMBD3. Our results provide insights into the function of AtMBD3 in plants, especially in sperm fertility.