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Background: Inappropriate pre-pregnancy body mass index (BMI) and gestational weight gain (GWG) are both linked to preterm birth (PTB); however, which one plays a dominant role in PTB risk is not yet sure. We aimed to evaluate the combined effect of pre-pregnancy BMI and GWG on the risk of PTB in singleton pregnancies conceived both spontaneously and through assisted reproductive technology (ART). Methods: The data included all mothers (n = 17,540,977) who had a live singleton birth from the US National Vital Statistics System (NVSS) 2015–2019. Logistic regression models, quantile-g-computation, and generalized additive model were used to analyze the combined association of pre-pregnancy BMI and GWG with PTB. Results: The singleton PTB rate was significantly higher in ART pregnancies (11.5%) than in non-ART pregnancies (7.9%). When compared to those women with pre-pregnancy normal weight and GWG within Institute of Medicine (IOM) guidelines, the highest PTB risk was observed in non-ART women with pre-pregnancy underweight and GWG below IOM guidelines (aOR 2.56; 95% CI 2.53–2.60) and in ART women with pre-pregnancy obese and GWG below IOM guidelines (aOR 2.56; 95%CI 2.36–2.78). GWG dominated the combined effect with its joint effect coefficient of − 0.281 (P < 0.05) in non-ART women and − 0.108 (P < 0.05) in ART women. Conclusions: Inappropriate GWG played a dominant role in increasing the risk of PTB in both non-ART and ART populations. Counseling regarding pre-pregnancy BMI and especially GWG appears to be even more crucial for pregnancies conceived via ART, given their impact on PTB. [ABSTRACT FROM AUTHOR]