To investigate the potential role of β-galactosidase in altering immunoglobulin G (IgG) galactosylation in serum of rheumatoid arthritis (RA). The expression level and activity of β-galactosidase in serum and CD 19 B cells were measured by enzyme-linked immune sorbent assay (ELISA). The effect of β-galactosidase on the -glycan changes in serum from mice intravenously treated with β-galactosidase was observed by linear ion-trap quadrupole-electrospray ionization mass spectrometry (LTQ-ESI-MS). We established a collagen-induced arthritis (CIA) rat model to explore the biological function of β-galactosidase in RA. The expression level of β-galactosidase in serum of 32 patients was elevated when compared with those of 30 healthy controls. The activity and expression level of β-galactosidase in CD19 B cells from RA patients was higher than those from healthy controls. The ratio of m/z 1142/937 was reduced in mice treated with β-galactosidase when compared with normal mice. We found that β-galactosidase was implicated in the development of inflammation by affecting body weight and elevating the expression level of interleukin-6， tumor necrosis factor-α， and rheumatoid factor in the serum. Our results suggested the high level of β-galactosidase in B cells and serum of RA patients and revealed that altered β-galactosidase may be implicated in the progression of inflammation.