Glucose-responsive insulin delivery systems that mimic pancreatic endocrine function could enhance health and improve quality of life for people with type 1 and type 2 diabetes with reduced β-cell function. However, insulin delivery systems with rapid in vivo glucose-responsive behaviour typically have limited insulin-loading capacities and cannot be manufactured easily. Here, we show that a single removable transdermal patch, bearing microneedles loaded with insulin and a non-degradable glucose-responsive polymeric matrix, and fabricated via in situ photopolymerization, regulated blood glucose in insulin-deficient diabetic mice and minipigs (for minipigs >25 kg, glucose regulation lasted >20 h with patches of ~5 cm2). Under hyperglycaemic conditions, phenylboronic acid units within the polymeric matrix reversibly form glucose-boronate complexes that-owing to their increased negative charge-induce the swelling of the polymeric matrix and weaken the electrostatic interactions between the negatively charged insulin and polymers, promoting the rapid release of insulin. This proof-of-concept demonstration may aid the development of other translational stimuli-responsive microneedle patches for drug delivery.