Prostate cancer (PCa) is a heterogeneous and unpredictable disease and becomes untreatable when the tumor progress to castrate-resistant (CR) or androgen independent (AI). A major clinical challenge in prostate cancer is the lack of diagnostic and prognostic tests that distinguish between different stages of the disease. Isoforms of gene transcripts are emerging as suitable candidates to represent disease progression. Vitamin D receptor transcript isoforms could be the target candidates of study since they have been related with anti-tumoral effects and carcinogenesis in several cancer types. The current study investigates the role of vitamin D receptor transcript isoforms in prostate cancer cell lines, PNT2, P4E6, LNCaP, DU145 and PC3; representing different progression and androgen dependency stages of PCa. Total RNA from these cell lines was sequenced with Illumina RNAseq Next Generation Sequencing (NGS) and expression values of the vitamin D receptors VDR, PDIA3 and RXRA; were analyzed. Absolute quantification of PDIA3 isoforms was performed with Droplet Digital PCR (ddPCR) in order to validate NGS findings. Functional and location prediction analysis of the different PDIA3 isoforms was performed with several bioinformatic tools. The NGS analysis revealed a novel PDIA3 transcript isoform (PDIA3N) that is higher expressed than the PDIA3 isoform that codifies for the receptor protein, in prostate cells. The expression of PDIA3N was validated by droplet digital PCR (ddPCR) absolute quantification, which confirmed the findings from the NGS analyses. The PDIA3N isoform was present in higher levels than PDIA3, in the metastatic androgen-sensitive LNCaP cells. Moreover, results shown that the ratio between PDIA3N and PDIA3 is related to androgen dependency and PCa progression. Finally, analysis of PDIA3N sequence indicate that the variations present in its sequence are altering the original protein function and structure as well as the predicted subcellular localization of the protein.We conclude that, PDIA3N due to the high expression in LNCaP cells and its abnormality in predicted structure, localization and function can be a potential target for the study of prostate cancer progression. The correlation of PDIA3N/PDIA3 ratio with PCa progression and androgen dependency stage will be further tested in PCa human samples.