The accurate callose deposition plays important roles in pollen wall formation and pollen fertility. As a direct target of miRNA160, ARF17 participate in the formation of the callose wall. However, the impact of ARF17 misexpression in microsporocytes on callose wall formation and pollen fertility remains unknown. Here, the SDS promoter, which is capable of specifically driving gene expression in microsporocytes, was employed to drive the expression of 5mARF17. The pSDS:5mARF17#3 transgenic line were male sterile. TEM revealed that sporopollenin substance was embedded in a thicker callose layer, which resulted in the complete loss of exine structure and pollen abortion in the pSDS:5mARF17#3 line. Consistently, RT-qPCR revealed an increase in the expression of several Cals genes in pSDS:5mARF17#3. EMSA assay demonstrated that ARF17 could bind to the promoter of Cals4 gene, which further suggest that ARF17 could regulate several Cals genes expression. It is notable that the expression of several exine formation-related genes increased significantly in pSDS:5mARF17#3. In conclusion, our findings highlight that the regulation of miRNA160-ARF17 in microsporocytes modulates the thickness of the callose wall, which is crucial for pollen exine formation and intercellular communication.