Mammary gland development and lactation are typical traits controlled by multiple genes, hormones, and regulatory factors. Prolactin receptor (PRLR), a specific receptor of prolactin, has been reported to have important physiological functions in regulating mammogenesis and lactogenesis. However, the post-transcriptional regulation mechanisms of PRLR expression have not yet been shown in detail. In this study, the expression of miR-135a and PRLR at different development stages of Laoshan dairy goat mammary gland tissues was investigated. After overexpression and silencing expression of miR-135a in cultured primary mammary epithelial cells, the regulatory relationship between miR-135a and PRLR was examined through dual-luciferase reporter assay, and the expression of PRLR at both mRNA and protein levels was examined by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Collectively, our results suggested that PRLR is a direct target gene of miR-135a, miR-135a is a novel regulator of PRLR, and it might play an essential role in the regulation of animal mammary gland development and lactation.