Brk (breast tumor kinase, also known as PTK6) is a nonreceptor tyrosine kinase that is aberrantly expressed in several cancers and promotes cell proliferation and transformation. Genome sequencing studies have revealed a number of cancer-associated somatic mutations in the Brk gene; however, their effect on Brk activity has not been examined. We analyzed a panel of cancer-associated mutations and determined that several of the mutations activate Brk, while two eliminated enzymatic activity. Three of the mutations (L16F, R131L, and P450L) are located in important regulatory domains of Brk (the SH3, SH2 domains, and C-terminal tail, respectively). Biochemical data suggest that they activate Brk by disrupting intramolecular interactions that normally maintain Brk in an autoinhibited conformation. We also observed differential effects on recognition and phosphorylation of substrates, suggesting that the mutations can influence downstream Brk signaling by multiple mechanisms.