Objective To explore the mechanism of enhanced pancreatic islet function in SD rats during middle to late pregnancy. Methods SD rats with pregnancy of 15 d were selected as experiment group (n=24), and another 24 rats of the same batch without pregnancy were served as controls (n=24). Single pancreatic islet was obtained by in situ perfusion and pancreatic islet isolation, pancreatic islet was digested by pancreatic enzyme, single cell was formed, isolated pancreatic islet was treated with DMEM culture medium containing 5.6 mmol/L glucose and 15% FBS. After culture with KRB buffer containing 2.8 mmol/L glucose (control) or 5.6 mmol/L glucose for 1 h, whole-cell patch-clamp was used to record pancreatic islet B cell membrane voltage-dependent potassium channel (Kv) current. Real-Time PCR was employed to detect the expression of prolactin receptor (PRLR), Janus tyrosine kinase 2 (JAK2), signal transducer and activator of transcription 5 (STAT5A and STAT5B), glucose transporter-2 (GLUT-2), glucokinase (GK), ATP-sensitive potassium channel (KATP) and voltage-dependent Ca²⁺ channel (VDCC) mRNA. Results Under the condition of 10 mV to 60 mV membrane voltage, Kv current of pancreatic islet B cell membrane in experiment group stimulated by 5.6 mmol/L glucose was significantly lower than that in control group (P<0.05). The relative expression of PRLR, JAK2, STAT5A, STAT5B, GLUT2, VDCC and KATP mRNA in tissues of pancreatic islet in experiment group was up-regulated, and the values were 2.50, 1.84, 1.54, 2.45, 1.41, 1.68 and 1.55 times of those in control group (P<0.05 or P<0.01). There was no significant difference in the relative expression of GK mRNA in tissues of pancreatic islet between two groups (P>0.05). Conclusion Lactogenic hormones play a primary role in pancreatic islet function enhancement during middle to late pregnancy in SD rats, which may be involved in the up-regulation of JAK2/STAT5 pathway, glucose metabolism and expression of insulin secretion-related molecules.