Litchi seed is a major by-product of the litchi processing industry, rich in polyphenols and flavonoids with documented anti-inflammatory properties. Prostatic inflammatory injury remains a prevalent male health concern, and dietary intervention with natural bioactive components represents a promising strategy for prostate health maintenance. This study investigated the protective efficacy of LCS2, a bioactive fraction isolated from litchi seed by-product, against lipopolysaccharide (LPS)-induced prostatic inflammatory injury in rats and explored its underlying mechanisms. LCS2 was obtained via extraction, purification, and activity screening, and its chemical profile was characterized by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). Its anti-inflammatory activity and pathway regulatory effects were evaluated in LPS-stimulated RAW264.7 macrophages and further verified in a rat model of LPS-induced acute prostatic inflammation. A total of 26 compounds, mainly flavonoids, polyphenols, and tannins, were tentatively identified in LCS2. In vitro, LCS2 pretreatment with LCS2 at concentrations up to 40 μg/mL dose-dependently suppressed LPS-induced overproduction of nitric oxide (NO) and reactive oxygen species (ROS), reduced pro-inflammatory cytokine mRNA levels, and downregulated iNOS and COX-2 protein expression. In vivo, 7-day dietary pretreatment with LCS2 at doses up to 100 mg/kg ameliorated prostate histopathological injury, reduced peripheral inflammatory cell counts, and decreased inflammatory mediator levels in serum and prostate tissue by inhibiting NF-κB and MAPK pathway activation. These findings support the high-value valorization of litchi seed by-product and highlight the potential of LCS2 for further development as a bioactive component for prostate health applications.