This study was conducted to test laser speckle perfusion imaging (LSPI) for imaging microcirculation and monitoring microcirculatory changes of port wine stains (PWS) during vascular targeted photodynamic therapy (V-PDT). Before and 5 min after V-PDT, PWS lesions and the corresponding contralateral healthy skins of 24 PWS patients were scanned, whereas seven PWS patients were scanned throughout V-PDT. V-PDT was conducted immediately after intravenous injection of photocarcinorin (4–5 mg kg−1). A 532 nm laser was used for irradiation (power density: 80–100 mW cm−2, exposure time: 20–50 min). Before V-PDT, all 24 PWS patients demonstrated a significant difference in perfusion between the PWS lesion and the contralateral healthy control skin (1132 ± 724 and 619 ± 478 PU, respectively, < 0.01). Five minutes after V-PDT, the mean perfusion value of the 24 PWS lesions was 1246 ± 754 PU. There was no significant difference compared to the perfusion before V-PDT (> 0.05). During V-PDT, the perfusion of seven PWS patients increased rapidly after initiation of V-PDT, reached a maximum within 10 min, lasted for several minutes, and slowly returned to a relatively lower level at the end of V-PDT. On the basis of these results, LSPI is capable of imaging PWS microvasculature and monitoring microvascular reactivity to V-PDT.