Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (eg, genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.