The human enteroviruses (HEV) are responsible for a large diversity of infections affecting humans. Most infections are asymptomatic, but these viruses can cause a wide spectrum of diseases, including severe cases involving the central nervous system. The aim of this study was to isolate and identify human enteroviruses in natural circulation in children less than 15 years of age admitted at the Mavalane General Hospital in Maputo City, Mozambique. In this study, 178 stool samples were processed, obtained during November 2011 to February 2012. Samples were inoculated onto cell culture and the isolates were identified as enterovirus by conventional RT-PCR in the 5’ non-coding region followed by partial VP1 sequence. Twenty-six (26) out of the 45 cell-culture positive samples were constituted by Enterovirus (14.6% of the total 178 samples). EV-29 was the serotype most prevalent. The results show the importance of maintaining the cell line Hep2C in the diagnosis and Enterovirus circulating in the Maputo city, Mozambique.