Background Acrosin binding protein (ACRBP) is a member of the cancer–testis antigen (CTA) family. Normally, ACRBP mRNA is expressed only in seminiferous tubules, while abnormally it is expressed in various types of cancers in tumor tissues, such as brain tumor. Objectives To determine the expression and clinical impact of a newly discovered splice variant of ACRBP in brain tumor. Methods Total RNA was extracted and reverse transcribed from 92 tumor specimens and 3 cell lines. Primers were designed to determine the expression of the new splice variant in all the samples. Quantitative real-time PCR (qPCR) was conducted for samples positive in reverse transcriptase-PCR. Association of the expression of ACRBP with the clinicopathological features of the various brain tumors was assessed statistically. Results The primers identified a newly discovered splice variant of ACRBP named ACRBP-V5a. The proportions of samples of the various brain tumor types positive for the ACRBP-V5a splicing variant were as follows: astrocytoma 10/33 (30%), glioblastoma 10/30 (33%), medulloblastoma 14/29 (48%), all tumors 34/92 (37%). Although we did not find a significant difference in the proportions of samples of various types of brain tumor tissues positive for the new splice variant (P > 0.05), levels of expression of the ACRBP-V5a splice variant were significantly different for tumor grade (P = 0.01) and tumor type (P = 0.02). Conclusions A newly discovered splice variant, ACRBP-V5a, is present in brain tumor. The new splicing variant may have discriminative value and potential importance in molecular-targeted therapy for brain tumors.