The membrane lipids from fast-elongating wild-type cotton (Gossypium hirsutum) fibers at 10 days post-anthesis, wild-type ovules with fiber cells removed, and ovules from the fuzzless-lintless mutant harvested at the same age, were extracted, separated, and quantified. Fiber cells contained significantly higher amounts of phosphatidylinositol (PI) than both ovule samples with PI 34:3 being the most predominant species. The genes encoding fatty acid desaturases (Δ(15)GhFAD), PI synthase (PIS) and PI kinase (PIK) were expressed in a fiber-preferential manner. Further analysis of phosphatidylinositol monophosphate (PIP) indicated that elongating fibers contained four- to five-fold higher amounts of PIP 34:3 than the ovules. Exogenously applied linolenic acid (C18:3), soybean L-α-PI, and PIPs containing PIP 34:3 promoted significant fiber growth, whereas a liver PI lacking the C18:3 moiety, linoleic acid, and PIP 36:2 were completely ineffective. The growth inhibitory effects of carbenoxolone, 5-hydroxytryptamine, and wortmannin were reverted by C18:3, PI, or PIP, respectively, suggesting that PIP signaling is essential for fiber cell growth. Furthermore, cotton plants expressing virus-induced gene-silencing constructs that specifically suppressed GhΔ(15)FAD, GhPIS, or GhPIK expression, resulted in significantly short-fibered phenotypes. Our data provide the basis for in-depth studies on the roles of PI and PIP in mediating cotton fiber growth.