Investigations on the inheritance and mechanism of resistance to Bacillus sphaericus Neide in Culex quinquefasciatus Say colonies, selected with strains C3-41 (RLCq1/C3-41) and 2362 (CqRL1/2362), were performed in China and Brazil, respectively. The progeny of reciprocal F1 crosses (susceptible female × resistant male and vice versa) from both resistant colonies responded alike in bioassays, indicating recessive inheritance. Data on larvae susceptibility from the backcross offspring between F1 and their respective susceptible and resistant parental colonies are consistent with a monofactorial and autosomal mode of inheritance. In vitro binding assays between ¹²⁵I binary (Bin2) toxin and the brush border membrane fractions (BBMF) from CqRL1/2362 and RLCq1/C3-41 larvae showed that resistance, in both colonies, is caused by a failure in the binding step of the B. sphaericus Bin2 toxin to its specific midgut receptor. The specific and saturable binding of Bin2 toxin to BBMF from F1 larvae (CqRL1/2362 X susceptible counterpart) confirms the recessive inheritance of the resistance gene. Further studies are needed to advance understanding of B. sphaericus resistance.