Colletotrichum falcatum, a hemibiotrophic fungal pathogen, causes one of the major devastating diseases of sugarcane-red rot. C. falcatum secretes a plethora of molecular signatures that might play a crucial role during its interaction with sugarcane. Here, we report the purification and characterization of a novel secreted protein of C. falcatum that elicits defense responses in sugarcane and triggers hypersensitive response (HR) in tobacco. The novel protein purified from the culture filtrate of C. falcatum was identified by MALDI TOF/TOF MS and designated as C. falcatum plant defense-inducing protein 1 (CfPDIP1). Temporal transcriptional profiling showed that the level of CfPDIP1 expression was greater in incompatible interaction than the compatible interaction until 120 h post-inoculation (hpi). EffectorP, an in silico tool, has predicted CfPDIP1 as a potential effector. Functional characterization of full length and two other domain deletional variants (CfPDIP1 Delta N1-21 and CfPDIP1 Delta N1-45) of recombinant CfPDIP1 proteins has indicated that CfPDIP1 Delta N1-21 variant elicited rapid alkalinization and induced a relatively higher production of hydrogen peroxide (H2O2) in sugarcane suspension culture. However, in Nicotiana tabacum, all the three forms of recombinant CfPDIP1 proteins triggered HR along with the induction of H2O2 production and callose deposition. Further characterization using detached leaf bioassay in sugarcane revealed that foliar priming with CfPDIP1a dagger 1-21 has suppressed the extent of lesion development, even though the co-infiltration of CfPDIP1a dagger 1-21 with C. falcatum on unprimed leaves increased the extent of lesion development than control. Besides, the foliar priming has induced systemic expression of major defense-related genes with the concomitant reduction of pathogen biomass and thereby suppression of red rot severity in sugarcane. Comprehensively, the results have suggested that the novel protein, CfPDIP1, has the potential to trigger a multitude of defense responses in sugarcane and tobacco upon priming and might play a potential role during plant-pathogen interactions.