Replication of tobacco mosaic virus (TMV) is connected with endoplasmic reticulum (ER)-associated membranes at early stages of infection. This study reports that TMV movement protein (MP)-specific protein kinases (PKs) associated with the ER of tobacco were capable of phosphorylating Thr¹⁰⁴ in TMV MP. The MP-specific PKs with apparent molecular masses of about 45–50 kDa and 38 kDa were revealed by gel PK assays. Two types of mutations were introduced in TMV MP gene of wild-type TMV U1 genome to substitute Thr¹⁰⁴ by neutral Ala or by negatively charged Asp. Mutation of Thr¹⁰⁴ to Ala did not affect the size of necrotic lesions induced by the mutant virus in Nicotiana tabacum Xanthi nc. plants. Conversely, mutation of Thr to Asp mimicking Thr¹⁰⁴ phosphorylation strongly inhibited cell-to-cell movement. The possible role of Thr¹⁰⁴ phosphorylation in TMV MP function is discussed.