The effects of microwave irradiation on the denaturation of collagen in acetic acid solution have been investigated in comparison with conventional heating. Circular dichroism measurements show that microwave irradiation can induce additional destructions of collagen triple helices due to nonthermal effect. Ultrasensitive microcalorimetry measurements further demonstrate the existence of nonthermal effect. The repeated exposure of collagen solutions to microwave along heating‐cooling cycles can only induce limited conformational changes measured with polarimetry. Our results indicate that thermal effect is the dominant factor leading to collagen denaturation, whereas nonthermal effect causes limited conformational destruction of the triple helices. PRACTICAL APPLICATION The thermal and nonthermal effects of microwave irradiation on biological system are still an open question. Whether nonthermal effect is associated with microwave irradiation and how it exerts influences if existing has no consistent statements so far. The present study helps to better understand the effect of thermal and nonthermal effects on the denaturation of protein upon microwave irradiation. The results suggest that microwave can be used in proteinous food processing safely because the total stereoisomers produced show no difference from conventional thermal denaturation of collagen.