The close association between rheumatoid arthritis (RA), sex, reproductive state, and stress has long linked prolactin (PRL) to disease progression. PRL has both proinflammatory and anti-inflammatory outcomes in RA, but responsible mechanisms are not understood. Here, we show that PRL modifies in an opposite manner the proinflammatory actions of IL-1β and TNF-α in mouse synovial fibroblasts in culture. Both IL-1β and TNF-α upregulated the metabolic activity and the expression of proinflammatory factors (Il1b, Inos, and Il6) via the activation of the nuclear factor-κB (NF-κB) signaling pathway. However, IL-1β increased and TNF-α decreased the levels of the long PRL receptor isoform in association with dual actions of PRL on synovial fibroblast inflammatory response. PRL reduced the proinflammatory effect and activation of NF-κB by IL-1β but increased TNF-α-induced inflammation and NF-κB signaling. The double-faceted role of PRL against the 2 cytokines manifested also in vivo. IL-1β or TNF-α with or without PRL were injected into the knee joints of healthy mice, and joint inflammation was monitored after 24 hours. IL-1β and TNF-α increased the joint expression of proinflammatory factors and the infiltration of immune cells. PRL prevented the actions of IL-1β but was either inactive or further increased the proinflammatory effect of TNF-α. We conclude that PRL exerts opposite actions on joint inflammation in males and females that depend on specific proinflammatory cytokines, the level of the PRL receptor, and the activation of NF-κB signaling. Dual actions of PRL may help balance joint inflammation in RA and provide insights for development of new treatments.