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Background: Human rabies cases in the Guangxi province of China decreased from 839 in 1982 to 24 in 1995, but subsequently underwent a sharp increase, and has since maintained a high level. Methodology/Principal Findings: 3,040 brain samples from normal dogs and cats were collected from 14 districts of Guangxi and assessed by RT-PCR. The brain samples showed an average rabies virus (RV) positivity rate of 3.26%, but reached 4.71% for the period Apr 2002 to Dec 2003. A total of 30 isolates were obtained from normal dogs and 28 isolates from rabid animals by the mouse inoculation test (MIT). Six representative group I and II RV isolates showed an LD50 of 10−5.35/ml to 10−6.19/ml. The reactivity of monoclonal antibodies (MAbs) to group I and II RV isolates from the Guangxi major epidemic showed that eight anti-G MAbs showed strong reactivity with isolates of group I and II with titers of ≥10,000; however, the MAbs 9-6, 13-3 and 12-14 showed lower reactivity. Phylogenetic analysis based on the G gene demonstrated that the Guangxi RV isolates have similar topologies with strong bootstrap values and are closely bonded. Alignment of deduced amino acids revealed that the mature G protein has four substitutions A96S, L132F, N436S, and A447I specific to group I, and 13 substitutions T90M, Y168C, S204G, T249I, P253S, S289T, V332I, Q382H, V427I, L474P, R463K Q486H, and T487N specific to group II, coinciding with the phylogenetic analysis of the isolates. Conclusions: Re-emergence of human rabies has mainly occurred in rural areas of Guangxi since 1996. The human rabies incidence rate increased is related with RV positive rate of normal dogs. The Guangxi isolates tested showed a similar pathogenicity and antigenicity. The results of phylogenetic analysis coincide with that of alignment of deduced amino acids. Author Summary: Rabies is a worldwide zoonosis disease and is of considerable public health threat and hazard. The Guangxi province of southern China is a severe rabies epidemic region. Human rabies cases decreased from 839 in 1982 to 24 in 1995 in Guangxi as a result of a dog vaccination campaign. However, the number subsequently underwent a sharp increase, and has since maintained a high level. This study reports the systematic surveillance of rabies in Guangxi over the 30-year period from 1982 to 2012. The data revealed that a re-emergence of human rabies has occurred mainly in rural areas of Guangxi since 1996. Human rabies incidence rate increased follows increased instances of RV positive normal dogs. To further understand this re-emergence of rabies, the biological properties of the rabies virus (RV), including the RV-positive rate of normal dogs, pathogenicity, antigenicity and evolution, have been evaluated. The Guangxi isolates all showed similar pathogenicity and antigenicity. These isolates also exhibited similar topologies with strong bootstrap values in the two groups and were closely bonded. Thus these findings will be helpful to understanding the epidemiological situation for rabies in Guangxi. [ABSTRACT FROM AUTHOR]