Metastatic castration-resistant prostate cancer (mCRPC) remains lethal, and lineage plasticity drives a subset of tumors to neuroendocrine prostate cancer (NEPC), most often treatment-emergent (t-NEPC). Prostate-specific membrane antigen (PSMA), the target of radioligand therapies for advanced prostate cancer (PCa), is heterogeneously expressed across metastatic sites but lost during neuroendocrine differentiation, excluding many patients from these therapies. The glycolytic enzyme Enolase 1 (ENO1) is a promising alternative target because it localizes to the surface of human tumors and promotes cancer growth and metastasis. However, it remains understudied in advanced PCa compared with ENO2 (neuron-specific enolase), a canonical neuroendocrine marker. We report here that total and cell surface ENO1 are more abundant than ENO2 in PSMA-negative mCRPC and NEPC cells. ENO1 silencing reduced cell proliferation and clonogenic growth even when triggering ENO2 upregulation. Targeting cell surface ENO1 with a monoclonal antibody decreased proliferation, clonogenicity, and tumorsphere formation in mCRPC cells, including docetaxel-resistant cells. This was associated with S-phase accumulation, elevated phospho histone p-H2A.X, and increased apoptosis. Significantly higher transcript expression of ENO1 than ENO2 was observed in clinical prostate tumors, including high-Gleason and neuroendocrine tumors, concomitant with elevated expression of their transcriptional regulator MYC. These findings position cell-surface ENO1 as a therapeutic and theranostic target for PSMA-low, treatment-refractory PCa.