Alpha-methylacyl-CoA racemase (AMACR) is a peroxisomal and mitochondrial enzyme involved in the beta-oxidation of branched fatty acids, shown to be elevated in prostate cancer by several recent studies. Sequence variants of AMACR have been linked to prostate cancer risk. Although mRNA transcript, protein, and sequence variants of AMACR have been studied in the context of prostate cancer, AMACR enzymatic activity has not been addressed. Here we present evidence that AMACR activity is consistently elevated in prostate cancer tissue specimens. This activity can be immunodepleted from prostate cancer tissue extracts. Furthermore, mock needle biopsy cores containing foci of prostate cancer exhibited increased AMACR enzymatic activity, correlating with both protein levels and histopathology. Taken together, our studies suggest that AMACR activity is increased in prostate cancer relative to benign epithelia and suggests that monitoring AMACR activity levels in prostate needle biopsies may have clinical applications.