This study investigated the effects of ambient cold exposure on inflammatory pain development, synovial cytokine levels, and spinal cord glial cell activation. Male Sprague–Dawley rats (6 weeks old) were divided into Cold and RT groups. The Cold group was exposed to cold (4 ± 1 °C) for 6 h/day for 5 consecutive days, while the RT group remained at room temperature (22 ± 1 °C). On day 6, knee arthritis was induced via intra-articular carrageenan injection. Pain was assessed by weight-bearing forces (WBFs) of the affected limb. Synovial pro-inflammatory (IL-1 β, IL-6, and TNF-α) and anti-inflammatory (IL-10) cytokines were measured by ELISA, while spinal cord microglia and astrocytes activation were evaluated via immunohistochemistry. WBFs were maximally reduced 4 h post-carrageenan injection, gradually recovering afterward. Cold-exposed rats showed significantly decreased WBF on days 1 and 2 post-injection compared to the RT group. In the Cold group, synovial cytokines (IL-1β, IL-6, IL-10) were significantly elevated 4 h post-injection, with no change in TNF-α levels. Additionally, OX42-positive cells (microglia) significantly increased 1 h post-injection in the Cold group, while GFAP-positive cells (astrocyte) remained unchanged. Repeated ambient cold exposure enhances inflammatory pain development through the regulation of synovial cytokines and microglia activation in the spinal cord in carrageenan-induced knee arthritis.