Monkeypox virus (MPXV), a member of the Orthopoxvirus genus, has begun to spread into many countries worldwide. While the prevalence of monkeypox in Central and Western Africa is well-known, the recent rise in the number of cases spread through intimate personal contact, particularly in the United States, poses a grave international threat. Previous studies have shown that cell-surface heparan sulfate (HS) is important for vaccinia virus (VACV) infection, particularly the binding of VACV A27, which appears to mediate the binding of virus to cellular HS. Some other glycosaminoglycans (GAGs) also bind to proteins on Orthopoxviruses. In this study, by using surface plasmon resonance, we demonstrated that MPXV A29 protein (a homolog of VACV A27) binds to GAGs including heparin and chondroitin sulfate/dermatan sulfate. The negative charges on GAGs are important for GAG–MPXV A29 interaction. GAG analogs, pentosan polysulfate and mucopolysaccharide polysulfate, show strong inhibition of MPXV A29–heparin interaction. A detailed understanding on the molecular interactions involved in this disease should accelerate the development of therapeutics and drugs for the treatment of MPXV.