Paeoniflorin (PF) has many effects, such as anti-inflammation, immune-regulation, abirritation, and so on. However, the protective mechanisms of PF on rheumatoid arthritis (RA) was not completely known. Thus, we explored deeply the protective mechanisms in a collagen-induced RA (CIA) rat model. CIA was induced in rats by intradermal injection of bovine type II collagen in complete Freund's adjuvant. Later, the CIA rats received oral administration of PF (50 and 100 mg/kg) once a day from the day 21, with the treatment lasting for 14 days. A variety of indicators were measured for evaluation of anti-rheumatism effect, including paw swelling, arthritis scores, and histopathological changes. And the contents of pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) in the serum, as well as p-NF-κB p65 and p-MYPT1 in the joint synovial tissues were detected to explore the possible mechanisms. The results demonstrated that PF treatment significantly ameliorated the symptoms in CIA rats, reduced the levels of pro-inflammatory cytokines and paw swelling, down-regulated the expressions of p-NF-κB p65 and p-MYPT1. The present results revealed that PF could effectively improve collagen-induced RA in rats by inhibiting Rho kinase activation in the joint synovial tissues, in turn down-regulating expression of p-NF-κB p65 and reducing contents of pro-inflammatory cytokines. Moreover, PF may be an effective agent for RA.