Prostate cancer is a major contributor to male mortality worldwide. In this study, we revealed that Ankyrin Repeat and SOCS Box Containing 1 (ASB1) expression was significantly decreased in prostate cancer tissues, correlating strongly with poor patient prognosis. Notably, the group with low ASB1 expression exhibited an increased proportion of M2 macrophages and showed resistance to immune checkpoint inhibitors and cisplatin, but remained sensitive to androgen-receptor-targeting drug bicalutamide. Silencing ASB1 enhanced prostate cancer cell proliferation, clonogenicity, and migration, whereas its overexpression exerted the opposite effects. Through quantitative mass spectrometry interactome analysis, we identified 37 novel proteins interacting with ASB1, including CHCHD3. Subsequent experiments including co-immunoprecipitation, cycloheximide treatment, and ubiquitination assays, revealed that ASB1 interacts with CHCHD3, promoting its degradation via K48-linked ubiquitination. Cell rescue experiments further demonstrated that ASB1 inhibits prostate cancer cell through the CHCHD3/reactive oxygen species (ROS) pathway. Taken together, our study indicated that ASB1 functions as a tumor suppressor by inhibiting CHCHD3/ROS signaling, thereby playing a vital part in prevention of prostate cancer proliferation, clonogenicity, and migration.