Herpes simplex virus type 1 H129 strain has been widely used as a useful anterograde neuronal circuit tracing tool. However, whether H129 is a rigorous anterograde tracer and undergoes anterograde-only spreading are questions of significant interest. In the present study, we evaluated the retrograde labeling efficiency of H129 using a TK and ICP34.5 dual deleted H129 recombinant (named as H306) which was replication-deficient in non-dividing postmitotic neurons. The novel tracer was tested in vitro and in vivo for evaluating its invasion properties and tracing capacities. The results demonstrated that H306 could efficiently label the neurons following intracerebral injection. Notably, H306 could also efficiently infect upstream innervating neurons through axon terminal uptake and displayed obvious retrograde labeling phenotype, regardless of 3 days or 10 days of tracing. The data implied that replication-competent, trans-multisynaptic H129 tracing results might be a mixed neural networks from two types of starter cells, because the retrogradely infected neurons would also replicate H129 and spread virus anterogradely through their axon collaterals (ectopic starter sites), as the local infected neurons in the injection site (true starter site). Therefore, the interpretation of the anterogradely tracing neural networks by current H129 tools at longer post-inoculation intervals need to be cautious, and effective modification strategies are needed to avoid or block the axon terminal invasion process of H129, which is important for rigorous anterograde H129 tracer.