The soybean cyst nematode (SCN, Heterodera glycines) relies on chemosensation for host localization and successful infection. Understanding the molecular basis of chemosensation is critical, as it determines the nematode’s ability to locate and invade host roots. In the model nematode Caenorhabditis elegans, Transient Receptor Potential Vanilloid (TRPV) channels osm-9 and ocr-2 and the G-protein α subunit goa-1 mediate sensory perception and ion transport; however, their roles in SCN remain largely unexplored. Here, we cloned and characterized these three candidate genes, which showed predominant expression in the phasmids of preparasitic juveniles. RNA interference revealed distinct roles: Hg-goa-1 regulated locomotion, whereas Hg-osm-9 and Hg-ocr-2 controlled chemotaxis, particularly attraction under acidic and basic conditions. Silencing any gene reduced root penetration and reproduction, while gene interaction analyses suggested a cross-regulatory network. Collectively, these results identify the three genes as essential regulators of SCN chemosensation, locomotion, and parasitism, providing potential molecular targets for developing species-specific nematicides.