The inflammatory response is a crucial physiological process that can lead to tissue damage and is considered a causative factor for various chronic diseases, such as rheumatoid arthritis. Recent research has focused on exploring valuable nutrients derived from Cannabis sativa L. (hemp) seeds, particularly hemp seed proteins. Therefore, this study aimed to investigate the release of anti-inflammatory peptides from Lactobacillus paraplantarum-fermented hemp seed proteins. To confirm the complete hydrolysis of hemp seed proteins during the fermentation process, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed. Further, the isolation and purification of peptides were achieved through ultrafiltration. The identity of peptides was nextly established using ultra-high performance liquid chromatography coupled with hybrid quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS). The results revealed a total of 39 identified peptides in fermented hemp seeds, with 9 peptides selected based on their relative quantity. Notably, AAELIGVP (P1), AAVPYPQ (P2), VFPEVAP (P4), DVIGVPLG (P6), and PVPKVL (P9) demonstrated strong anti-inflammatory abilities in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells. Molecular docking was used to understand the potential anti-inflammatory mechanism of these 5 peptides, and in silico results indicated that P1, P2, P4, P6, and P9 could bind to the active sites of toll-like receptor 4 (TLR-4), nuclear factor-κB (NF-κB), and inhibitor of NF-κB kinase (IKK) with higher binding energies. Overall, these findings indicate that hemp seeds have potential to be a source of bioactive peptides for functional foods with anti-inflammatory properties.