Objective To isolate and identify the proteins that interact with cardiac troponin I (cTnI) in primary mouse cardiomyocytes, and to analyze their biological processes and signaling pathways by use of High throughput proteomics and bioinformatics techniques. Methods The hearts of C57 neonatal mice were used to prepare the primary cardiomyocytes. The cTnI binding proteins were extracted from the whole cell protein by co-immunoprecipitation, and the proteins were analyzed and identified by mass spectrometry. Furthermore, the physicochemical properties, biological processes and signaling pathways of cTnI interacting proteins were predicted by bioinformatics tools. Results A total of 262 proteins were identified by mass spectrometry, cTnI binding proteins were involved in 14 biological processes and 33 KEGG biological pathways (P<0.05). Conclusion In mouse cardiomyocytes, 262 kinds of proteins may interact with cTnI, and participate in regulation of calcium ion influx and PI3K-Akt signaling pathways. DOI: 10.11855/j.issn.0577-7402.2017.09-04