<p>A large number of extrachromosomal circular DNAs (eccDNAs) are found in eukaryotic cells, but the mechanism behind the formation of eccDNAs remains unknown. EccDNAs smaller than 1000 bp are commonly referred to as microDNAs. In a prior study, we identified a 542 bp microDNA, designated eccDNA^fib-L, mapping to Chromosome 14: 9,692,083-9,692,624 nt in the silk gland of <i>Bombyx mori</i>. There is a direct short repeat “GAGT” at both 5′ and 3′ break points of eccDNA^fib-L, but only one copy of “GAGT” is retained in eccDNA^fib-L. Here, we find that the specific junction observed can also be detected by transfecting with a DNA fragment containing upstream sequences of the 5′ break point and downstream sequences of the 3′ break point of eccDNA^fib-L. Additionally, the length of the flanking sequences and the direct short repeats “GAGT” affect the formation efficiency of eccDNA^fib-L. We confirm that eccDNA^fib-L formation is associated with DNA repair pathways, with the expression of eccDNA^fib-L undergoing significant alterations following the silencing of genes related to DNA repair pathways. In vitro, a cell-free reaction system confirms that Polθ and the direct short repeat “GAGT” are essential for the circularization of eccDNA^fib-L, and the DExH-box helicase domain of Polθ plays critical role in mediating this circularization process. Collectively, our data support a mechanism whereby Polθ can mediate the joining of linear DNA fragments with direct short repeat at the 5′ and 3′ ends to form a circular DNA through MMEJ. The results not only clarify the essential factors driving eccDNA^fib-L formation but also complement previous reports on eccDNA formation.</p><p></p>