Background: Pathogenesis of aortic stenosis (AS) involves lipid infiltration, inflammation, and oxidative stress, which drive calcification of the aortic valve and progression to heart failure (HF). Fatty acids (FAs) play a crucial role in these processes. A treatment option for severe symptomatic AS in elderly and high-risk patients is transcatheter aortic valve implantation (TAVI). Objective: To investigate the change in FA profiles in patients undergoing TAVI. Methods: This single-center prospective study included 25 patients with severe AS qualified for TAVI procedure. Blood samples were collected before TAVI and after six months. FA profiles were analyzed by gas chromatography-electron ionization mass spectrometry. Results: Notable changes were identified in FA profiles, including a reduction in docosahexaenoic acid (DHA) levels (117 ± 48.0 µM vs. 141 ± 53.0 µM, p = 0.001) and an increase in alpha-linolenic acid (ALA) concentration (32.8 ± 12.3 µM vs. 19.9 ± 6.40 µM, p = 0.003) six months post-TAVI. Additionally, significant elevations were noted in specific medium-chain FAs (C12) and branched-chain fatty acids (iso C16, iso C17 and anteiso C15, anteiso C17) at six months after TAVI. However, total n-3 polyunsaturated fatty acids (n3 PUFA) levels decreased (p = 0.039), while n-6 polyunsaturated fatty acids (n6 PUFA) levels exhibited no significant overall change at this time point. Decrease in mean pressure gradient (PG) was negatively correlated with eicosapentaenoic acid (EPA), DHA, n-3 docosapentaenoic acid (DPA n3) and n3 PUFA levels in a six-month observation. Conclusions: Our results underscore the complex interplay between cardiac intervention and FA changes, providing novel insights into the metabolic impact of TAVI on FAs serum profile.