Rotator cuff tendinopathy is a common musculoskeletal disorder with limited pharmacological treatment strategies. This study aimed to investigate tenocytes’ functional in vitro response from a ruptured supraspinatus tendon to suramin administration and to elucidate whether suramin can enhance tendon repair and modulate the inflammatory response to injury. Tenocytes were obtained from human supraspinatus tendons (n = 6). We investigated the effect of suramin on LPS-induced inflammatory responses and the underlying molecular mechanisms in THP-1 macrophages. Suramin enhanced the proliferation, cell viability, and migration of tenocytes. It also increased the protein expression of PCNA and Ki-67. Suramin-treated tenocytes exhibited increased expression of COL1A1, COL3A1, TNC, SCX, and VEGF. Suramin significantly reduced LPS-induced iNOS, COX2 synthesis, inflammatory cytokine TNF-α production, and inflammatory signaling by influencing the NF-κB pathways in THP-1 cells. Our results suggest that suramin holds great promise as a therapeutic option for treating rotator cuff tendinopathy.