The aim of the present study was to optimize niosomes by experimental design for enhanced transdermal delivery of ursolic acid for the effective treatment of arthritis. The experimental design (3 factor 3 levels, Box–Behnken design) was used to study individual and combined effects of different formulation variables. The variables cholesterol (X1), span 60 (X2) and phospholipid (X3) were taken as independent factors and their effect was observed on size (Y1) entrapment efficiency (Y2), and transflux (Y3). The formulation composition with span 60 (85 mg), cholesterol (12.3 mg), and phospholipid (65 mg) was found to fulfil requisites of optimized ursolic acid niosome formulation (URNF). URNF had shown vesicle size of 665.45 nm, entrapment efficiency of 92.74% with transflux of 17.25 μg/cm2/h. The in vivo bioactivity showed that the prepared URNF-gel was able to provide good anti-arthritic activity due to enhanced permeation of UA through the skin and results were found to be comparable to standard gel (Omni gel). The radiographical image confirmed that, the developed URNF-gel was found to be effective to treat arthritis. Thus niosomal gel of ursolic acid would be a promising alternative to conventional therapy for safe and efficient treatment of arthritis and musculoskeletal disorders.