Abstract Background Auxin conjugates are hydrolyzed to release free auxin to ensure defined cellular auxin levels or gradients within tissues for proper development or response to environmental signals. The auxin concentration in the abscission zone (AZ) is thought to play an important role in mediating the abscission lag phase. Results In this study, the full cDNA sequences of seven tomato ILR1-like SlILL genes were identified and characterized, All SlILLs were found to have auxin conjugate hydrolysis activity. The effects of different auxin conjugates on abscission identified IAA-Ile as a candidate to determine the auxin conjugate and auxin conjugate hydrolysis functions in abscission. Treatment of pedicel explants with IAA-Ile for different times showed that application before 6 h could effectively delay abscission. IAA-Ile pre-incubation for 2 h was sufficient to inhibit abscission. These results showed that there is not sufficient auxin conjugates in the AZ to inhibit abscission, and the optimal time to inhibit abscission by the application of exogenous auxin conjugates is before 6 h. Treatment with cycloheximide (CHX, a protein biosynthesis inhibitor) indicated that de novo synthesis of auxin conjugate hydrolases is also required to delay abscission. During abscission, SlILL1, 5, and 6 showed abscission-related gene expression patterns, and SlILL1, 3, 5, 6, and 7 showed increasing expression trends, which collectively might contribute to delay abscission. Silencing the expression of SlILL1, 3, 5, 6, and 7 using virus-induced gene silencing showed that SlILL1, 5, and 6 are major mediators of abscission in tomato. Conclusions In the process of abscission, auxin inhibition is concentration dependent, and the concentration of auxin in the AZ was regulated by hydrolyzed auxin conjugates. SlILR1, 5, and 6 play a key role in flower pedicel abscission.