κ-opioid receptor (κ-OR) plays a key role in preventing hypoxic pulmonary hypertension (HPH) development after activated by exogenous agonist U50,488H. Calcium sensing receptor (CaSR) activation induces HPH by promoting vasoconstriction and vascular remodeling. The activated κ-OR is reported to inhibit the expression of CaSR in pulmonary artery smooth muscle cells (PASMCs). Thus, in this study, we aimed to explore the effect of activated κ-OR on the role of CaSR in preventing HPH development. An HPH rat model was constructed using Sprague-Dawley rats. Changes in mean pulmonary arterial pressure (mPAP) and right ventricular pressure (RVP) mediated by κ-OR agonist U50,488H and CaSR inhibitor NPS2143 were observed. The effects of CaSR agonist spermine and inhibitor NPS2143 on pulmonary artery tension were tested. The expression and localization of κ-OR and CaSR were measured in isolated PASMCs. A cell-counting kit-8 assay was performed to evaluate the effect of spermine in PASMC proliferation. Expression of proliferating cell nuclear antigen (PCNA), Erk, and p-Erk was evaluated by western blot analysis. Results showed that κ-OR and CaSR were co-expressed and colocalized in PASMCs under normoxic and hypoxic conditions. Interactions between κ-OR and CaSR were also observed. Spermine improved vasoconstriction in the pulmonary artery in HPH rats, which was abolished by U50,488H. RVP and mPAP were significantly increased in HPH rats under CaSR stimulation, but were significantly reduced when the rats were pretreated with U50,488H and NPS2143 (P < 0.01). Spermine treatment significantly promoted PASMC proliferation, which was significantly inhibited by U50,488H, p38 inhibitor SB203580, JNK inhibitor SP600125, Erk inhibitor SCH772984, and MEK inhibitor U0126, especially Erk inhibitor (P < 0.01). Spermine significantly increased PCNA and P-Erk expression in hypoxic conditions, which was inhibited by U50,488H and NPS2143. κ-OR stimulation prevented HPH development via the CaSR/MAPK signaling pathway.