Worldwide measurements of nearly 130 C₃ species covering all major plant functional types are analysed in conjunction with model simulations to determine the effects of mesophyll conductance (g_m) on photosynthetic parameters and their relationships estimated fromA/C_i curves. We find that an assumption of infinite g_m results in up to 75% underestimation for maximum carboxylation rate V_cmax, 60% for maximum electron transport rate J_max, and 40% for triose phosphate utilization rate T_u. V_cmax is most sensitive, J_max is less sensitive, and T_uhas the least sensitivity to the variation of g_m. Because of this asymmetrical effect of g_m, the ratios of J_max to V_cmax, T_u to V_cmax and T_u toJ_max are all overestimated. An infinite g_m assumption also limits the freedom of variation of estimated parameters and artificially constrains parameter relationships to stronger shapes. These findings suggest the importance of quantifying g_m for understanding in situphotosynthetic machinery functioning. We show that a nonzero resistance to CO₂ movement in chloroplasts has small effects on estimated parameters. A non-linear function with g_m as input is developed to convert the parameters estimated under an assumption of infinite g_m to proper values. This function will facilitate g_m representation in global carbon cycle models.