The heart is capable of adapting to various environmental stresses by flexibly changing its structure. Hemodynamic overload (or mechanical stress) and cardiotoxic drugs, such as doxorubicin, cause pathological cardiac remodeling in which abnormal production of reactive oxygen species (ROS) plays a critical role. We have recently showed that functional coupling between transient receptor potential canonical (TRPC) 3 and NADPH oxidase 2 (Nox2) mediated aberrant ROS production during pathological remodeling of mouse hearts. In this study, we found that extracellular ATP caused atrophy of neonatal rat cardiomyocytes (NRCMs), which was attenuated by silencing TRPC3 or Nox2 genes. ATP increased expression of muscle atrophy-related E3 ubiquitin ligase, MAFbx. TRPC3 and Nox2 were responsible for ATP-induced ROS production and atrophy of NRCMs. We demonstrated that ATP treatment caused physical interaction between TRPC3 with Nox2 in TRPC3 and Nox2-overexpressing HEK293 cells. The ATP-induced increase of TRPC3-Nox2 interaction was also observed in NRCMs as well. In summary, ATP may induce cardiomyocyte atrophy through increases in MAFbx expression and ROS production by activating TRPC3-Nox2 functional coupling.