Reduced prolactin (PRL) has been shown to delay wound healing with a limited understanding of the underlying mechanisms. Here, we aim to explore the role of PRL in the repair of vocal fold (VF) injury. A microarray was used to detect the expressed levels of PRL in rat VF tissue at 1, 4, and 8 weeks after VF injury compared with normal uninjured rats. Then, a systematic bioinformatics analysis has been conducted to explore the literature-based biology network and signaling pathways involved in the repair of VF injury. The expression of PRL was significantly decreased in all VF injury groups (week 1, 4, and 8) compared with the control group (F stats = 280.34; P=4.88e-14), with no significant difference among the three VF injury groups (F stats = 1.97; P=0.18). Wounding has been shown to interfere with both PRL-promoting and inhibiting pathways that were involved in wound healing, including 11 PRL inhibitors and 6 PRL promoters. Our results reveal decreased PRL expression levels in VF injury, which is not in favor of the wound healing. The pathways identified may help in understanding the role of PRL as a treatment target for VF wound healing.