Introduction. Phototherapy is gaining increased attention in the research and treatment of various diseases. In particular, the use of blue light seems to bear promise, owing to its antimicrobial and immune-modulating properties; however, research focused on the effects of blue light on keratinocytes and reepithelization is rare. In addition, few studies to date have evaluated devices that are used in daily hospital routine. Objective. This study investigated the effects of phototherapy on keratinocytes with 2 established devices in vitro. Materials and Methods. Human adult low calcium high temperature keratinocytes were irradiated with 2 different devices, and the effects on scratch wound closure, proliferation, cell viability, and cytokine expression were evaluated. Results. Blue light irradiation reduced reepithelization at high doses in a scratch wound healing model (wound closure on day 1: control group, 25.57 percentage points [PP] ± 2.36 standard deviation vs Device A for 10 minutes, 1.33 PP ± 1.01) and mitochondrial activity measured with resazurin conversion (Device A for 10 minutes, 33.28% ± 12.34). Irradiated cells demonstrated a lower ratio of proliferating cell nuclear antigen-positive cells and, as a result, lower proliferation. Conclusions. Blue light reduces keratinocyte proliferation and migration at high doses and therefore could negatively affect wound healing. Available irradiation devices for possible use in wound therapy should be critically scrutinized and evaluated with in vitro methods prior to clinical use.