Molecular crowding in cells alters all binding affinities. Spatial differences in cytosolic composition differentially affect crowding properties regulating attachment and release of molecules and/or molecular complexes. Actin polymerization in cell occurs in a variety of places that may be differentially affected by effects of molecular crowding. There are also specific effects exerted by different crowding agents. We found that a naturally occurring osmolyte trimethylamine N-oxide (TMAO) dramatically increases affinities of intrinsically disordered actin-regulatory proteins thymosin beta 4 (TB4) and thymosin beta 10 to ATP-G-actin and significantly decreases barbed end actin critical concentration Ac in presence of ATP. On the other hand, effect of TMAO on the affinity to ATP-G-actin of a globular actin regulating protein profilin is much weaker.Interestingly, the dependence of the amount of sequestered actin in presence of TB4 is bell-shaped. The standard free energy change depends non-linearly on TMAO concentration for Ac and linearly for the affinity of TB4 to actin. This implies that spatial variation in intracellular conditions can exert complex effects on actin sequestration.We also found that the TMAO facilitates a ternary complex formation between actin, profilin, and TB4 which may very significantly increase the amount of unpolymerized actin in presence of profilin and TB4 and slow down actin polymerization. Our results support the hypothesis that regulation of actin polymerization could be spatially regulated by modulation of the effects of natural osmolytes in different locations inside the cells. Moreover, the data suggest that a potential explanation for the perplexing question as to why these two actin-binding proteins, profilin and thymosin are well-structured and disordered, respectively. We speculate that the compactly structured profilin can impart a structural change to actin while disordered thymosin may create a mechanism for regulation of the ternary complex by crowding effects.