Background This study investigated the activation of mitogen‐activated protein kinases in the spinal dorsal horn to explore the mechanisms underlying morphine‐induced acute and chronic hyperalgesia in mice. Methods Male adult mice were given a single subcutaneous injection (SC) of morphine (1 μg/kg) or twice‐daily administration of morphine (10 mg/kg/day) for 8 days. Thermal hyperalgesia and mechanical allodynia were assessed using the radiant heat and von Frey filament test. Levels of phospho (p)‐extracellular signal–regulated kinases (p‐ERK), p‐c‐Jun N‐terminal kinase (p‐JNK), p‐p38, p‐PKCγ, N‐methyl‐d‐aspartate receptor (NMDAr), and c‐Fos protein in the spinal dorsal horn were examined by Western blot assays. Results A single ultra‐low dose or repeated administration of morphine induced hyperalgesia in mice and caused a significant increase in the levels of p‐ERK and p‐JNK, but not p‐p38, in the spinal dorsal horn. The level of c‐Fos protein was significantly elevated following administration of morphine. The protein levels of p‐PKCγ and NMDAr subunits (NR2B and NR2A) were also altered. Pretreatment with the NMDAr antagonist MK‐801 or the protein kinase C (PKC) inhibitor calphostin C (CC) suppressed the morphine‐induced increase in p‐ERK, p‐JNK, and c‐Fos. Administration of MK‐801 and CC also relieved morphine‐induced hyperalgesia. Conclusion These findings suggest that activation of the spinal ERK and JNK signaling pathways contribute to morphine‐induced acute and chronic hyperalgesia in mice. In this study, we evaluated our findings which suggest the activation of the spinal extracellular signal–regulated kinases, and the c‐Jun N‐terminal kinase signaling pathway contributes to morphine‐induced acute hyperalgesia in mice.