Pregnancy induces significant physiological changes to support foetal growth, including critical adaptations in pancreatic islets for glucose homeostasis. This study elucidates these adaptations in human islets using immunohistochemistry, with a focus on α- and β-cell metrics, prolactin receptor (PRLR), and serotonin 2B receptor (5-HT2B) expression during pregnancy. Utilising formalin-fixed paraffin-embedded (FFPE) pancreatic tissue sections from pregnant and non-pregnant donors, we employed laser capture microdissection (LCM) to isolate islets followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for proteomic analysis. We observed a 1.9-fold increase in islet area, with α-cell and β-cell areas expanding by 4.3-fold and 1.9-fold, respectively, driven by cell number rather than hypertrophy. Notably, PRLR expression was upregulated in α-cells but not β-cells, and 5-HT2B receptors were absent in β-cells. Proteomic profiling revealed minimal changes in protein expression, suggesting nuanced islet adaptations during pregnancy. Key differentially expressed proteins included cathepsin Z, cyclin-dependent kinase 5, and laminin subunit alpha 4. Our findings highlight the distinct molecular mechanisms of human islets compared to rodent models, underscoring the necessity of human-based studies for advancing understanding of islet adaptations in pregnancy.