The c-Abl tyrosine kinase maintains cellular homeostasis through its ability to regulate apoptosis and actin dynamics. In vivo, c-Abl activity is stringently regulated and mechanisms involved are not fully understood. Here, we identified the Rap1 guanine nucleotide exchange factor, C3G (RapGEF1), as a substrate and an effector of c-Abl-mediated functions. Ectopic expression of c-Abl in mammalian cell lines, known to induce apoptosis, resulted in phosphorylation of endogenous C3G on Y504 coincident with cell detachment and chromatin condensation. Phosphorylation of C3G coincided with restricted c-Abl activation in regions rich in actin, and was dependent on cellular F-actin dynamics. Unlike C3G or c-Abl, p-C3G was resistant to detergent extraction, suggesting its enhanced affinity for the cytoskeleton. Localized C3G phosphorylation and coincidence with cells undergoing cell death was dependent on F-actin-binding domain (FABD) of c-Abl. Activation of endogenous c-Abl by oxidative stress was associated with phosphorylation of cellular C3G on Y504. Inhibition of C3G expression and function using RNAi or dominant-negative approaches inhibited c-Abl-mediated cell death. These findings identify C3G as a novel target of c-Abl and also show that FABD of c-Abl is essential for regulation of its restricted activation to induce apoptosis.