The microRNAs (miRNAs) are a class of small non-coding RNA molecules that play important roles in cellular processes by regulating gene expression at the post-transcriptional level. In skin biology, several miRNAs have been shown to be associated with differentiation, migration and apoptosis of keratinocytes, and regulation of the hair cycle. Although the biological role of miR-330-5p has been reported in several cancers and cells, the function and molecular mechanism of miR-330-5p in skin keratinocytes have not been identified. In this study, we found that miR-330-5p inhibited the proliferation and migration of mouse keratinocytes. Among the candidate target genes of miR-330-5p searched using microarray analysis, we found that the expression of Pdia3 was directly regulated by miR-330-5p in the mouse keratinocyte. Moreover, inhibition of Pdia3 expression caused decreased proliferation and migration ability of mouse keratinocytes. Additionally, expressions of miR-330-5p and Pdia3 displayed an inverse correlation with respect to the hair cycle stage. These results indicated that regulation of Pdia3 expression by miR-330-5p is important in maintaining the hair cycle through regulation of the proliferation and migration capability of keratinocytes.