While mammary tumours are the main reasons of death in bitches, early detection of tumours and metastases is crucial for survival of affected dogs. Invasiveness and angiogenesis, which are important processes of tumour growth and spreading, require connective tissue remodelling. This process is dominantly mediated by matrix metalloproteinases (MMP), which are well known to be positively regulated by relaxin (RLX) in various tissues, including human breast cancer. So far, the presence of RLX and its receptor RXFP-1 as well as their linkage with MMP in canine mammary tumours (CMT) is completely unknown. In the first part of the present study, concentrations of RLX, oestradiol and progesterone from plasma samples of bitches with CMT were compared with clinical and survival data to investigate the predictive value of these hormones. In the second part, the expressions of RLX, RXFP-1 and MMP-2, -9 and -13 were examined by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in 31 CMT samples. Finally, relationships of systemic plasma RLX or locally expressed RLX with expression of MMP in CMT were analyzed for the first time. Comparison of hormone concentrations in 93 bitches in terms of benign or malignant nature of the CMT, lung metastases, recidivation and 12-month survival discovered no significances. The expressions of RLX, RXFP-1 and MMP were independent from plasma RLX, but expressions of local RLX and RXFP-1 showed a strong correlation (p = 0.00004, r = 0.671) as well as RXFP-1 and MMP-2 (p = 0.009, r = 0.463), indicating a possible significant role of the locally produced RLX in CMT pathogenesis as an inducer of connective tissue remodelling.