To investigate the effect of acupuncture on progesterone (P4) and prolactin (PRL) in rats of embryo implantation dysfunction (EID). On the first day of pregnancy, 72 female Wistar rats were randomly allocated into the normal group, the EID model group, the acupuncture group and the P4 group (18 in each group). The normal group was injected sesame oil, while the other three groups were given mifepristone to establish the EID model. The acupuncture group and the P4 group were given treatment of acupuncture and P4 injection, respectively. The serum of P4 and PRL were detected by radioimmunoassay, and the mRNA and protein expressions of P4 receptor (PR) and PRL receptor (PRLR) were detected using real-time polymerase chain reaction and immunohistochemical method, respectively. Compared with the normal group, the serum levels of P4 and PRL as well as the mRNA and protein expression levels of PR and PRLR in the EID model group were significantly lowered (P < 0.01 or P < 0.05). The above indices in the acupuncture group and the P4 group were significantly elevated compared with the EID model group (P < 0.01 or P < 0.05). Acupuncture can promote embryo implantation effectively, which might be related to the effects of acupuncture on upregulating the P4 and PRL levels in serum and the PR and PRLR expression levels in rats.