To analyze the epidemiological and etiological characteristics of Hand-Foot-and-Mouth disease (HFMD) in Shanghai in 2009, epidemiological data was retrieved from the National Notifiable Disease Report System (NNDRS). Nucleic acid of enterovirus (EV) was detected by real-time RT-PCR from 799 HFMD cases from 15 districts/counties in Shanghai; the complete sequences of VP1 encoding region of several identified EV71 strains and sequences of VP4 encoding region of several untyped EV were determined and analyzed. Analysis and summary of the epidemiological data was conducted with Microsoft Excel, and sequence analyses were conducted with both BioEdit and MEGA software. Untyped EV was identified through comparing the VP4 sequence to sequence database using BLAST online service. It was showed that all the 18 districts/counties had reported HFMD cases; children less than 6 years old were the most susceptible population group; the peak of epidemics of HFMD was from April to July; EV71 and Coxsackievirus A16 (CA16) were the major pathogens for this epidemic, but the constituent ratio of EV71 and CA16 was different in different months and regions; CA16 infection was mainly responsible for the mild HFMD, but EV71 for most of the severe cases; EV71 strains of Shanghai were clustered with representatives of subgenotype C4a and showed the highest identity to them, based on the sequence analyses of VP1 encoding region; 2 of the untyped EV were identified as CA2 and CA10 respectively. All the results indicated that EV71 and CA16 were the major pathogens for the epidemic of HFMD in Shanghai, 2009; the circulating EV71 belonged to subgenotype C4a. Besides, other types of EV (for example: CA2 and CA10) were also responsible for a few of the HFMD cases.