At the 3′-end of telomeres, single-stranded G-overhang telomeric repeats form a stable T-loop. Many studies have focused on the mechanisms that generate and regulate the length of telomere 3′ G-strand overhangs, but the roles of G-strand overhang length control in proper T-loop formation and end protection remain unclear. Here, we examined functional relationships between the single-stranded telomere binding protein GTBP1 and G-strand overhang lengths maintained by telomerase in tobacco (Nicotiana tabacum). In tobacco plants, telomerase reverse transcriptase subunit (TERT) repression severely worsened the GTBP1 knockdown phenotypes, which were formally characterized as an outcome of telomere destabilization. TERT downregulation shortened the telomere 3′ G-overhangs and increased telomere recombinational aberrations in GTBP1-suppressed plants. Correlatively, GTBP1-mediated inhibition of single-strand invasion into the double-strand telomeric sequences was impaired due to shorter single-stranded telomeres. Moreover, TERT/GTBP1 double knockdown amplified misplaced homologous recombination of G-strand overhangs into intertelomeric regions. Thus, proper G-overhang length maintenance is required to protect telomeres against intertelomeric recombination, which is achieved by the balanced functions of GTBP1 and telomerase activity.