The effect of inbreeding and gene action on butternut squash (Cucurbita moschata Duch.) seed production and seed starch content was evaluated at two locations in the department of Valle del Cauca, Colombia, using six accessions (S0) and their inbred lines S1 and S2. Significant differences were found between the accessions and inbred lines, but not between localities. The seed production and seed starch content showed no significant effects of inbreeding depression. An additive-type gene action predominated, suggesting that recurrent selection could be the best strategy to increase the frequencies of genes promoting seed production and seed starch content.