Picornavirus infection involves a dynamic interplay of host and viral protein interactions that modulates cellular processes to facilitate virus infection and evade host-antiviral defenses. Here, using a proteomics-based approach to identify protease-generated neo-N-termini peptides, we identify a novel target of the poliovirus 3C proteinase, the heterogeneous nuclear ribonucleoprotein M (hnRNP M), which is a nucleo-cytoplasmic shuttling RNA-binding protein that is primarily known for its role in pre-mRNA splicing. hnRNP M is cleaved in vitro by poliovirus and coxsackievirus B3 (CVB3) 3C proteinases and is targeted in poliovirus- and CVB3-infected HeLa cells and in hearts of CVB3-infected mice. hnRNP M relocalizes from the nucleus to cytoplasm during poliovirus infection. Finally, depletion of hnRNP M using siRNA knockdown approaches decreases poliovirus and CVB3 infections in HeLa cells and does not affect poliovirus IRES translation and viral RNA stability. We propose that cleavage of and subverting the function of hnRNP M is a general strategy utilized by picornaviruses to facilitate viral infection. IMPORTANCE: Enteroviruses, a member of the picornavirus family, are RNA viruses that cause a range of diseases including respiratory ailments, dilated cardiomyopathy and paralysis. Although enteroviruses have been studied for several decades, the molecular basis of infection and the pathogenic mechanisms leading to disease are still poorly understood. Here, we have identified hnRNP M as a novel target of a viral proteinase. We demonstrate that the virus subverts the function of hnRNP M and redirects it to a step in the viral life cycle. We propose that cleavage of hnRNP M is a general strategy that picornaviruses use to facilitate in infection.