The calpain family, and their endogenous inhibitor calpastatin, has been implicated in cancer progression, and recent in vitro data have indicated a role in trastuzumab resistance. The aims of our study were to examine expression levels of calpastatin, calpain-1 and calpain-2 in breast tumours from patients treated with trastuzumab following adjuvant chemotherapy to determine their potential as biomarkers to predict therapeutic response. The expression of calpastatin, calpain-1 and calpain-2 was determined, using immunohistochemistry (IHC), in tumours from a series of 93 patients with primary breast cancer treated with surgery and adjuvant chemotherapy with or without trastuzumab followed by trastuzumab to complete 1 year of therapy. IHC was performed using tissue microarrays constructed from cores taken from intratumour and peripheral tumour areas. Expression was correlated with clinicopathologic variables and patient outcome. Calpastatin expression was correlated with Nottingham prognostic index (p = 0.003) and lymph node status (p = 0.007). Trastuzumab resistance was defined as disease relapse during therapy. Calpain-1 expression is associated with relapse-free survival (p = 0.001) and remained significant in multivariate analysis accounting for confounding pathological and treatment variables (hazard ratio 4.60, 95% confidence interval 1.05–20.25; p = 0.043). Calpain-1 may be a useful biomarker to predict relapse-free survival in breast cancer patients treated with adjuvant trastuzumab and chemotherapy. A larger verification study is warranted.