Accumulating evidence suggests that synovitis is associated with osteoarthritic process. Macrophages play principal role in development of synovitis. Our earlier study suggests that interaction between cartilage fragments and macrophages exacerbates osteoarthritic process. However, molecular mechanisms by which cartilage fragments trigger cellular responses remain to be investigated. Therefore, the current study aims at analyzing molecular response of macrophages to cartilage fragments. To this end, we analyzed the transcriptional profiling of murine macrophages exposed to cartilage fragments by RNA sequencing. A total 153 genes were differentially upregulated, and 105 genes were down-regulated in response to cartilage fragments. Bioinformatic analysis revealed that the most significantly enriched terms of the upregulated genes included scavenger receptor activity, integrin binding activity, TNF signaling, and toll-like receptor signaling. To further confirm our results, immunohistochemical staining was performed to detected regulated molecules in synovial tissues of OA patients. In consistence with RNA-seq results, MARCO, TLR2 and ITGα5 were mainly detected in the intima lining layer of synovial tissues. Moreover, blockade of TLR2 or ITGα5 but not Marco using specific antibody significantly reduced production of TNF-α in stimulated macrophages by cartilage fragments. Our data suggested that blocking TLR2 or ITGα5 might be promising therapeutic strategy for treating progressive osteoarthritis.