In our continuing efforts to find novel anti-HIV compounds, we have synthesized sixteen novel pyrazolo[4,3-c]pyridin-4-one derivatives. All the synthesized compounds were screened for anti-HIV activity against HIV-1VB59 (R5, subtype C). Compounds 12a-12c and 12e were also tested against HIV-1UG070 (X4, subtype D) in TZM-bl cell line. Compound 12c was found to be the most active against HIV-1VB59 and HIV-1UG070 with IC50 value 3.67 μM and 2.79 μM, and therapeutic indices 185 and 243, respectively. The lead compound 12c inhibited the HIV-192/BR/018 (R5, subtype B) and drug resistant isolates, NIH-119 (X4/R5, subtype B) and NARI-DR (R5, subtype C) effectively. The activity of the lead compound was further confirmed by PBMC assays. The molecular docking data showed that the most active compound 12c binds in the non-nucleoside binding pocket of HIV-1 reverse transcriptase, which was confirmed by the ToA assay. Thus the study indicated that 12c may be considered as a NNRTI and further explored as a lead for anti-HIV drug development.