In many plant species, petal abscission can be considered the final step of petal senescence. Cytokinins (CKs) are powerful suppressors of petal senescence; however, their role in petal abscission is ambiguous. Here, we observed that, in rose (Rosa hybrida), biologically active CK is accumulated during petal abscission and acts as an accelerator of the abscission process. Using a combination of reverse genetics, and molecular and biochemical techniques, we explored the roles of a LESION SIMULATING DISEASE1 (LSD1) family member RhLOL1 interacting with a bHLH transcription factor RhILR3 in CK-induced petal abscission. Silencing RhLOL1 delays rose petal abscission, while the overexpression of its ortholog SlLOL1 in tomato (Solanum lycopersicum) promotes pedicel abscission, indicating the conserved function of LOL1 in activating plant floral organ abscission. In addition, we identify a bHLH transcription factor, RhILR3, that interacts with RhLOL1. We show that RhILR3 binds to the promoters of the auxin signaling repressor auxin/indole-3-acetic acid (Aux/IAA) genes to inhibit their expression; however, the interaction of RhLOL1 with RhILR3 activates the expression of the Aux/IAA genes including RhIAA4-1. Silencing RhIAA4-1 delays rose petal abscission. Our results thus reveal a RhLOL1-RhILR3 regulatory module involved in CK-induced petal abscission via the regulation of the expression of the Aux/IAA genes.