2021/05/21 | Roasso Kumamoto | | 3-0 | | Nippon Bunri University | | | | |
2021/05/22 | Ehime FC | | 1-2 | | Imabari | | | | |
| Nagano Parceiro | | 3-1 | | Niigata UHW | | | | |
| Honda Lock | | 5-0 | | Kawasoe Club | | | | |
| Mitsubishi Mizushima | | 3-1 | | Baleine Shimonoseki | | | | |
| Arterivo Wakayama | | 3-3 (4-5 Pen.) | | Ococias Kyoto | | | | |
| Gifu | | 0-2 | | Honda FC | | | | |
| Sagamihara | | 3-1 | | Komazawa University | | | | |
| Iwaki | | 2-2 (2-4 Pen.) | | Sony Sendai | | | | |
| Ryutsu Keizai Univ. | | 2-3 | | YSCC | | | | |
| Renofa Yamaguchi | | 1-1 (2-4 Pen.) | | Verspah Oita | | | | |
2021/05/23 | Kataller Toyama | | 8-0 | | Hokuriku University | | | | |
| Porvenir Asuka | | 0-2 | | FC Osaka | | | | |
| Vanraure Hachinohe | | 13-0 | | Saruta Kogyo | | | | |
| Fukuyama City | | 2-1 | | Kagura Shimane | | | | |
| Fukui United | | 2-1 | | Aventura Kawaguchi | | | | |
| Suzuka Point Getters | | 2-1 | | FC Kariya | | | | |
| Iwate Grulla Morioka | | 13-0 | | Oyama SC | | | | |
| Blaublitz Akita | | 1-1 (8-9 Pen.) | | Hokkaido Tokachi | | | | |
| Biwako Seikei Sport College | | 0-2 | | Kwansei Gakuin University | | | | |
| Kagoshima United | | 4-0 | | MD Nagasaki | | | | |
| Fukuoka University | | 1-3 | | Okinawa SV | | | | |
| Gainare Tottori | | 4-1 | | FC Tokushima | | | | |
| Kochi United | | 1-0 | | Kamatamare Sanuki | | | | |
2021/05/26 | Tochigi City | | 9-0 | | Nirasaki Astros | | | | |
2021/06/05 | Tonan Maebashi | | 1-6 | | Juntendo University | | | | |