Description: Purpose: Enzalutamide (ENZ) is a potent androgen receptor (AR) antagonist with activity in castration-resistant prostate cancer (CRPC); however, progression to ENZ-resistant (ENZ-R) CRPC frequently occurs with rising serum PSA levels, implicating AR full-length (AR FL ) or variants (AR-Vs) in disease progression. Experimental Design: To define functional roles of AR FL and AR-Vs in ENZ-R CRPC, we designed 3 antisense oligonucleotides (ASO) targeting exon-1, intron-1, and exon-8 in AR pre-mRNA to knockdown AR FL alone or with AR-Vs, and examined their effects in three CRPC cell lines and patient-derived xenografts. Results: ENZ-R-LNCaP cells express high levels of both AR FL and AR-V7 compared with CRPC-LNCaP; in particular, AR FL levels were approximately 12-fold higher than AR-V7. Both AR FL and AR-V7 are highly expressed in the nuclear fractions of ENZ-R-LNCaP cells even in the absence of exogenous androgens. In ENZ-R-LNCaP cells, knockdown of AR FL alone, or AR FL plus AR-Vs, similarly induced apoptosis, suppressed cell growth and AR-regulated gene expression, and delayed tumor growth in vivo . In 22Rv1 cells that are inherently ENZ-resistant, knockdown of both AR FL and AR-Vs more potently suppressed cell growth, AR transcriptional activity, and AR-regulated gene expression than knockdown of AR FL alone. Exon-1 AR-ASO also inhibited tumor growth of LTL-313BR patient-derived CRPC xenografts. Conclusions: These data identify the AR as an important driver of ENZ resistance, and while the contributions of AR FL and AR-Vs can vary across cell systems, AR FL is the key driver in the ENZ-R LNCaP model. AR targeting strategies against both AR FL and AR-Vs is a rational approach for AR-dependent CRPC. Clin Cancer Res; 21(7); 1675–87. ©2015 AACR .