Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein‐C (cMyBP‐C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca²⁺ sensitivity of force (pCa₅₀), PKA treatment has been shown to decrease pCa₅₀, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca²⁺‐independent force and maximum Ca²⁺‐activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase‐specific differential effects on steady‐state force, we used synchrotron low‐angle X‐ray diffraction to compare equatorial intensity ratios (I_1,1/I_1,0) to assess the proximity of myosin cross‐bridge mass relative to actin and to compare lattice spacings (d_1,0) to assess the inter‐thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP‐C increases I_1,1/I_1,0 and, as hypothesized, treatment with MLCK also increased I_1,1/I_1,0, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by ∼2 nm (Δ 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP‐C phosphorylation increases the proximity of cross‐bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP‐C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca²⁺ sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.