Self-incompatibility (SI) is a genetic system that promotes outcrossing by rejecting self-pollen. In the Brassicaceae the SI response is mediated by the pistil S-locus receptor kinase (SRK) and its ligand, pollen Slocus cysteine-rich (SCR) protein. Transfer of SRK-SCR gene pairs to self-fertile Arabidopsis thaliana enabled establishment of robust SI, making this transgenic self-incompatible A. thaliana an excellent platform for SI analysis. Here we report isolation of a novel A. thaliana self-incompatibility mutant, AtC24 SI mutant, induced by heavy-ion beam irradiation. We show that the AtC24 SI mutant exhibits breakdown of SI, with pollen hydration, pollen tube growth and seed set resembling the corresponding processes in wild-type (self-fertile) A. thaliana. Further reciprocal crosses indicated that some perturbed SI factor in the stigmatic cell of the AtC24 SI mutant is responsible for the observed phenotype, while the pollen response remained intact. Our results demonstrate successful application of heavy-ion beam irradiation to induce a novel A. thaliana self-incompatibility mutant useful for SI studies.